Uteroglobin therapy for epithelial cell cancer

ABSTRACT

The invention relates to method for identifying prostatic intraepithelial neoplasia, methods for determining metastatic potential of tumors, and to methods and compositions for inhibiting or preventing metastasis of cancers. In one aspect, the invention provides a method to determine metastatic potential of tumors, particularly prostatic tumors. In another aspect, the invention provides a method of identifying prostate cancer associated conditions, particularly prostatic intraepithelial neoplasia. In these regards, the invention relate to determining protein or mRNA of effectors of arachidonic acid release, particularly uteroglobin protein or mRNA, to identify intermediate conditions such as PIN or to gauge metastatic potential of prostatic tumors. 
     The invention also relates to methods and compositions that prevent or inhibit metastasis of cancers. In this regard, the invention particularly relates to methods and compositions that inhibit arachidonic acid, those that inhibit phospholipase A 2 . More particularly in this regard, the invention relates to uteroglobin or muteins, peptide analogs or mimetics of uteroglobin and lipocortins or muteins, peptide analogs, or mimetics of lipocortins that inhibit metastasis. Especially it relates to methods and compositions in which uteroglobins, particularly human uteroglobins, inhibit or prevent metastasis of cancer, particularly prostatic cancer.

This application is a divisional of U.S. patent application Ser. No.08/966,196, filed Nov. 7, 1997 now U.S. Pat. No. 6,054,320, which is adivisional of U.S. patent application Ser. No. 08/658,796, filed Jun. 5,1996, issued as U.S. Pat. No. 5,935,860 on Aug. 10, 1999, which is a CIPof U.S. patent application Ser. No. 08/486,203, filed Jun. 7, 1995,issued as U.S. Pat. No. 5,830,640 on Nov. 3, 1998, which is a CIP ofU.S. patent application Ser. No. 08/400,084, filed Mar. 7, 1995, issuedas U.S. Pat. No. 5,696,092 on Dec. 9, 1997.

The present invention relates to methods and compositions that providefor the diagnosis and treatment of prostatic intraepithelial neoplasia.A particular aspect of the invention relates to methods and compositionscontaining compounds which inhibit phospholipase A₂, particularly thosethat contain uteroglobin, uteroglobin muteins, uteroglobin mimetics,peptide analogs of uteroglobins, lipocortins, lipocortin muteins andpeptide analogs of lipocortins. Further compositions of the inventioninclude other types of active ingredients in combination with thosedescribed above.

The present invention also relates to methods for gauging the metastaticpotential of tumors of epithelial cell origin by determining an effectorof arachidonic acid release in cells of a tumor-containing tissue. Thisaspect of the invention particularly relates to determining uteroglobinprotein or mRNA in cells of a biopsy sample to determine metastaticpotential of a prostatic tumor.

The present invention further relates to methods and compositions thatprevent or inhibit metastases of cancers of epithelial cell origin,especially human prostate cancers. A particular aspect of the inventionrelates to methods and compositions that inhibit arachidonic acidrelease in cells of these cancers and inhibit or prevent metastasis. Inone aspect in this regard, the invention particularly relates to methodsand compositions that inhibit phospholipase A₂ that mediates arachidonicacid release in the cancer cells. Compositions of the invention alsoparticularly include those that contain uteroglobin, uteroglobinmuteins, peptide analogs of uteroglobins, lipocortins, lipocortinmuteins and peptide analogs of lipocortins that inhibit arachidonic acidrelease by cancer cells. Further useful in this regard are mimeticcompounds, particularly uteroglobin and lipocortin mimetics. In thisregard, the invention relates especially to compositions that containmimetics of uteroglobin, particularly of human uteroglobin. Furthercompositions of the invention include other types of active ingredientsin combination with those that inhibit arachidonic acid release.

The invention also particularly relates to methods to prevent or inhibitmetastases of human cancers of epithelial cell origin by administeringthe foregoing compositions. Especially in this regard the inventionrelates to methods using human uteroglobin to inhibit or preventmetastasis of human prostate cancers. Further, this aspect of theinvention may be accomplished by genetic therapy.

Methods and compositions of the invention may be used by themselves andwith other treatment modalities.

BACKGROUND OF THE INVENTION

Cancers develop from uncontrolled multiplication of cells. All cancersare life threatening. Even when cancer does not result in death, it ispermanently debilitating, not only to the patient, but also to family,friends and co-workers. Too often, moreover, cancers prove fatal. Thepersonal and public loss from this cluster of diseases, which cause asignificant fraction of all premature deaths, is beyond estimation.

Although effective treatment modalities have been developed in a fewcases, many cancers remain refractory to currently available therapies.Particularly difficult to treat are metastatic cancers. These cancerspose the highest risk to patients and, for optimal prognosis, often mustbe treated by aggressive methods that present increased risks ofdeleterious side-effects. Therefore, there is a great need for methodsthat accurately distinguish those tumors that are likely to metastasizefrom those that are unlikely to do so. Furthermore, methods for treatingmetastatic cancers often are inadequate, and there also is a clear needfor improved anti-metastatic agents and methods to treat metastaticcancers.

Similarly, there is a great need for methods that accurately identifycells that are associated with prostate cancer, such as those found inprostatic intraepithelial neoplasia (PIN). Current diagnostic methodsare inadequate to differentiate between PIN and normal cells. Thus,there is a clear need for improved early detection of PIN which mayallow for early diagnosis, prognosis, and treatment of cancer.

Metastatic cancers originate from a primary tumor. Metastasis of theprimary tumor produces secondary tumors and disseminated cancer. It iswell known that both primary and secondary tumors shed large numbers ofcells. The shed cells can spread through the body. For instance, aprimary tumor may damage the surrounding lymph or circulatory vessels,allowing entry of shed cells into the lymph or circulatory systems, andhastening their spread in the body. Moreover, shedding of cells bycancerous tumors increases during surgery and radiotherapy.

Most shed cells do not form new tumors. To do so such cells mustsurmount a series of physical and physiological barriers. In fact, aseries of distinct events must occur for metastasis to occur. Theprimary tumor physically must (i) invade interstitial space of theprimary tissue. In particular, it must (ii) penetrate the basementmembrane of the tissue. For most metastases the tumor must damage theendothelial cell wall of lymphatic or vascular vessels to provide accessto shed cells. Cells that enter the lymph or blood must (iii) survivehemodynamic stress and host defense in the circulation and, furthermore,(iv) the cells must lodge at a new site in the circulatory system, aprocess that apparently involves aggregated platelets. A cell then must(v) extravasate out of the vessel into the interstitial space. Finally,it must (vi) invade the interstitial space of the secondary organ andproliferate in the new location. Although the process of metastasis isphysiologically complex, the overall pattern of metastasis is general tomany types of cancers.

The metastatic process also clearly involves complex intracellularmechanism that alter cancerous cells and their interactions withsurrounding cells and tissues. For instance, cancerous cells arecharacterized by aberrant expression of adhesion proteins, enzymes thatdegrade matrix components, autocrine factors, ligand-responsivereceptors, factors of angiogenesis and prostaglandins, to name a few. Inparticular, the signaling pathways that initiate tumor cell migrationare among the least understood aspects of invasion and metastasis.Currently, it is thought that proliferation of many cancerous cellsdepends upon specific ligand-receptor interactions. Thus far, however,it has not been possible to use this paradigm, or other concepts of theunderlying mechanisms of metastasis, to develop a therapy that preventsor effectively inhibits metastasis of metastatic cancers.

The complexity of the processes involved in metastasis, and the lack ofunderstanding of underlying molecular mechanisms, have made itparticularly difficult, in some cases, to distinguish tumors that arelikely to metastasize from those that are unlikely to do so. Theinability to discern the metastatic potential of tumors precludesaccurate prognosis and leads, inevitably, to the therapeuticintervention that either is too aggressive or insufficiently aggressive.Furthermore, for all types of cancers it has been difficult orimpossible, thus far, to develop treatments that inhibit or prevent thespread of metastatic tumors. Clearly, these remains a great need formethods to accurately determine the metastatic potential of tumors andfor effective anti-metastatic compositions and methods.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide methodsand compositions for differentiating PIN from normal prostate epithelia.

It is also an object of the present invention to provide methods andcompositions for early detection of prostate cancer and cells associatedwith prostate cancer.

It is another object of the present invention to provide methods forinhibiting or preventing metastasis.

It is another object of the present invention to provide compositionsfor inhibiting or preventing metastasis.

In accomplishing the foregoing objects, there has been provided, inaccordance with one aspect of the present invention, a method foridentifying prostatic intraepithelial neoplasia, comprising the step ofadministering to an organism suffering from a cancer of epithelial cellorigin a compound that inhibits arachidonic acid release by cells of thecancer by a route and in an amount and manner effective to identify theprostatic intraepithelial neoplasia.

Another method of the present invention is directed to a diagnostic kitfor the detection of prostatic intraepithelial neoplasia in a biopsysample, the kit comprising: a first reagent that binds specifically toan effector of arachidonic acid release in cells in a biopsy sampleprepared for determination of the effector, and a second reagent fordetectably labelling the primary binding reagent bound specifically tocells in the biopsy sample, wherein the determination of the effector isdiagnostic of prostatic intraepithelial neoplasia.

In certain preferred embodiments of the kits of the invention, theeffector is an inhibitor of PLA₂, among which uteroglobin isparticularly preferred.

In certain further preferred embodiments of the this aspect of theinvention, the first reagent is an antibody. In these embodiment, thedetermination would occur where uteroglobin-antibody staining wouldindicate normal prostate epithelia if strong staining occurred,prostatic intraepithelial neoplasia if weak staining occurred, andcancer if no signal occurred.

Additional preferred embodiments of this aspect of the invention arethose in which the first reagent is a hybridization probe. In certainpreferred embodiments of this aspect of the invention, the effector isan inhibitor of PLA₂, among which uteroglobin is particularly preferred.

Another preferred embodiment of the present invention is directed to amethod for preventing or inhibiting metastasis of a cancer of epithelialcell origin, comprising the step of administering to an organismsuffering from a cancer of epithelial cell origin a compound thatinhibits arachidonic acid release by cells of the cancer by a route andin an amount effective to inhibit or prevent metastasis of the tumor.

In a preferred embodiment of an aspect of the invention in this regard,the compound is an inhibitor of phospholipase A₂ or cyclooxygenase.Particularly, phospholipase A₂ inhibitors are preferred.

In certain particularly preferred embodiments of this aspect of theinvention, the compound is a uteroglobin, a mutein of a uteroglobin, apeptide analog of a uteroglobin, a mimetic of uteroglobin, a lipocortin,a mutein of a lipocortin, a peptide analog of a lipocortin or a mimeticof lipocortin. Especially highly preferred in this regard are methodswherein the compound is a uteroglobin, a mutein of a uteroglobin, apeptide analog of a uteroglobin or a mimetic of uteroglobin. Uteroglobinis preferred and human uteroglobin is particularly highly preferred inthis regard.

Also there is provided in accordance with this aspect of the inventioncertain preferred embodiments in which the compound is a small moleculedrug that is a nonsteroidal anti-inflammatory agent. Among these agentsinhibitors of phospholipase A₂ and cyclooxygenase are preferred.Particularly preferred are mepacrine and indomethacin.

In another regard preferred embodiments of the present method are thoseused to treat a cancer of the prostate gland in a human patient.

In further preferred embodiments, the method is used in conjunction withanother treatment. In this regard, preferred treatments include surgicalintervention, radiation therapy, hormonal therapy, immunotherapy,chemotherapy, cryotherapy or gene therapy.

In accordance with another aspect of the present invention, there hasbeen provided a pharmaceutical composition for inhibiting or preventingmetastasis of a cancer of epithelial cell origin, comprising: (i) acompound that inhibits arachidonic acid release by cells of a tumor ofepithelial cell origin effective to inhibit or prevent metastasis of thetumor in an organism and (ii) a carrier for effective the therapeuticadministration of the compound to the organism.

In certain preferred embodiments of the invention the compound is aninhibitor of phospholipase A₂ or cyclooxygenase. In this regard,inhibitors of phospholipase A₂ are preferred. In certain particularlypreferred embodiments the compound is a uteroglobin, a mutein of auteroglobin, a peptide analog or a uteroglobin, a mimetic ofuteroglobin, a lipocortin, a mutein of a lipocortin a peptide analog ofa lipocortin or a mimetic of lipocortin. In especially preferredembodiments in this regard the compound is a uteroglobin, a mutein of auteroglobin or a peptide analog of a uteroglobin. Among these,uteroglobins are very highly preferred and human uteroglobins are amongthe most highly preferred compounds of the present invention.

Also there is provided in accordance with this aspect of the inventioncertain preferred embodiments in which the compound is a small moleculedrug that is a nonsteroidal anti-inflammatory agent. Among these agentsinhibitors of phospholipase A₂ and cyclooxygenase are preferred.Particularly preferred are mepacrine and indomethacin.

In accordance with another aspect of the invention there has beenprovided a method for determining metastatic potential of a tumors,particularly those of epithelial cell origin. In certain preferredembodiments of this aspect of the invention there has been provided amethod for determining the metastatic potential of tumors of epithelialcell origin comprising the steps of (A) determining an effector ofarachidonic acid release in cells in a biopsy sample of a tumor; (B)comparing effector in tumor cells in the biopsy sample with effector infiduciary cells, and (C) determining metastatic potential, whereineffector in the tumor cells characteristic of normal fiduciary cells orcharacteristic of fiduciary cells of benign tumors indicates lowmetastatic potential and effector in the tumor cells characteristic offiduciary cells of metastatic tumors indicates high metastaticpotential.

In some preferred embodiments of this aspect of the invention theeffector is an inhibitor of PLA₂. In particularly preferred embodimentsin this regard, the effector is uteroglobin.

In certain preferred embodiments the effector is determined by assayingthe effector protein in cells of the tumor. In particularly preferredembodiments in this regard, the effector is an inhibitor of PLA₂.Especially preferred is uteroglobin. In particularly preferredembodiments in this regard the tumor is a prostatic tumor and theinhibitor is uteroglobin.

In another aspect of the invention, preferred embodiments of theinvention provide methods for determining metastatic potential in whicha protein is assayed by immunocytochemistry. In certain preferredembodiments of this type, the effector is an inhibitor of PLA₂.Particularly preferred in embodiments of the invention in this regard isuteroglobin. In particularly preferred embodiments in this regard thetumor is a prostatic tumor and the inhibitor is uteroglobin.

In certain additional preferred embodiments of the invention in thisregard, the effector is determined by assaying an mRNA in cells of atumor. In particularly preferred embodiments in this regard, the mRNAencodes an inhibitor of PLA₂. Especially preferred is uteroglobin. Inparticularly preferred embodiments in this regard the tumor is aprostatic tumor and the inhibitor is uteroglobin.

In certain preferred embodiments in this regard, the mRNA is determinedby a method comprising a step of hybridizing a probe to cells fixed on asurface. In certain preferred embodiments of this aspect of theinvention the mRNA is determined by in situ hybridization.

In preferred embodiments of the invention in both regards the effectoris an inhibitor of PLA₂, most particularly uteroglobin. In particularlypreferred embodiments in this regard the tumor is a prostatic tumor andthe inhibitor is uteroglobin.

In another aspect of the invention in this regard, aberrant mRNA isdetermined. In preferred embodiments of the invention in this regard,the mRNA encodes an inhibitor of PLA₂, most particularly uteroglobin. Inparticularly preferred embodiments in this regard the tumor is aprostatic tumor and the inhibitor is uteroglobin.

In a still further object of the invention there has been provided a kitfor determining metastatic potential of a tumor. In certain preferredembodiments kits of the invention comprise: (A) a first reagent thatbinds specifically to an effector or arachidonic acid release in cellsin a biopsy sample prepared for determination of the effector, and (B) asecond reagent for detectably labelling the primary binding reagentbound especially to cells in the biopsy sample, wherein thedetermination of the effector tumor is diagnostic of the metastaticpotential of the tumor.

In certain preferred embodiments of the kits of the invention, theeffector is an inhibitor of PLA₂, among which uteroglobin isparticularly preferred.

In certain further preferred embodiments of the this aspect of theinvention, the first reagent is an antibody. In particularly preferredembodiments in this respect, the effector is an inhibitor of PLA₂, amongwhich uteroglobin is particularly preferred.

Additional preferred embodiments of this aspect of the invention arethose in which the first reagent is a hybridization probe. In certainpreferred embodiments of this aspect of the invention, the effector isan inhibitor of PLA₂, among which uteroglobin is particularly preferred.Other objects, features and advantages of the invention will be apparentfrom the following detailed description. It should be understood,however, that the detailed description and the specific examples, whileproviding general and specific descriptions and indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of theinvention will become apparent to those skilled in the art from thedetailed description and other aspects of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar chart showing the dose effect of uteroglobin on theinvasiveness of cells of the TSU-PR1 and DU-145 cell lines, epithelialcell lines derived from human prostate tumors. Both TSU-Pr1 and DU-145cells are androgen independent. Cells were cultured in zero, 0.01, 0.1and 1.0 μM uteroglobin for 24 hr. Invasiveness then was assayed bymigration through filters coated with reconstituted basement membrane(RBM) in response to serum free or fibroblast conditioned media (FCM).Invading cells were stained with crystal violet, the dye was extractedand invasiveness was determined by measuring dye concentration byoptical absorbance at 585 nm. Each point in the graph is the mean ofresults of three separate experiments, each carried out using triplicatecultures. The bars show the standard error of the mean for each point.

FIG. 2 is a bar chart showing the dose effect of uteroglobin on theinvasiveness of cells of the PC3-M and LNCaP cell lines, epithelial celllines derived from human prostate tumors. LNCaP cells areandrogen-sensitive. PC3-M cells are androgen independent. Assays wereperformed as described in the caption to FIG. 1.

FIG. 3 is a bar chart showing that myoglobin, albumin andheat-inactivated uteroglobin do not affect the invasiveness of DU-145cells. Assays were performed essentially as described in the caption toFIG. 1.

FIG. 4 is a graph showing a time course of the effect of uteroglobin oninvasiveness of FCM-stimulated DU-145 cells. Cells were incubatedwithout or with 1.0 μM uteroglobin for 3, 5, 12, or 24 hr. and thenassayed for invasion in response to FCM as described in the caption toFIG. 1.

FIG. 5 is a graph showing that uteroglobin inhibits arachidonic acidrelease from FCM-stimulated DU-145 cells over a five hour period. Cellswere incubated for 24 hr. in αMEM/SF media containing ¹⁴C-labelledarachidonic acid. Free label then was washed away and the cells wereincubated in FCM without and with 1 μM uteroglobin. Arachidonic acidrelease was measured by ¹⁴C released from the cells into the medium.

GLOSSARY

The abbreviations and terms in the present disclosure are employed incontemplation of their fullest meaning consistent with the disclosed andclaimed invention. The following brief explanations are entirelyillustrative and neither exhaustively define nor limit the inventiondisclosed and claimed herein. The full meaning of the terms will beclear from an understanding of the invention based on contemplation ofthe disclosure as a whole in light of a full understanding of thepertinent arts.

ARACHIDONIC ACID CASCADE; a series of enzymatic reactions that resultsin the production and release of arachidonic acid by a cell. The cascadeis sensitive to ligand signals and arachidonic acid itself is anautocrine factor.

DU-145: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

LNCaP: an epithelial cell line derived from a human prostate tumor,which is androgen sensitive. The LNCaP cell line was derived from asupraclavicular lymph node metastasis of a human prostate carcinoma.Cells of this line exhibit increased proliferation in response toandrogen, in vitro, and they secrete prostate specific antigen (PSA), amarker of differentiated epithelial cells.

TSU-Pr1: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

PC3-M: an epithelial cell line derived from a human prostate tumor,which is androgen independent.

EFFECTOR: a substrate that engenders, alters, modulates or controls anactivity of a cell; a substance that can engender, alter, modulate orcontrol a physiological activity of a cell or organism. Typically, aprotein, such as an enzyme, cofactor or transcription regulatoryprotein, or an activator or inhibitor of an enzyme, an enzyme complex, areceptor or a receptor complex, for instance.

EPITHELIAL CELL ORIGIN: derived from an epithelial cell, of whatevertissue.

FCM: fibroblast conditioned media

FIDUCIARY: a reference against which a test outcome is compared to gaugeresults. A fiduciary series is a plurality of such references thatrepresent points along a qualitative or a quantitative scale.

IDENTIFYING: in the context of identifying PIN, ascertaining,establishing or otherwise determining one or more factualcharacteristics of prostatic intraepithelial neoplasia or a similarintermediate condition.

INHIBITION: inhibition of metastasis may be measured by many parametersin accordance with the present invention and, for instance, may beassessed by delayed appearance of secondary tumors, slowed developmentof primary or secondary tumors, decreased occurrence of secondarytumors, slowed or decreased severity of secondary effects of disease,arrested tumor growth and regression of tumors, among others. In theextreme, complete inhibition, is referred to herein as prevention.

METASTASIS: As set out in Hill, R. P., Chapter 11, Metastasis, pp178-195 in The Basic Science of Oncology, Tannock et al., Eds.,McGraw-Hill, New York (1992), which is incorporated by reference hereinin its entirety, metastasis is “The ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (i.e., toform metastases).”

Similarly, metastasis is described in Aznavocrian et al., Cancer 71:1368-1383 (1993), which is incorporated by reference herein in itsentirety, as “The transition from in situ tumor growth to metastaticdisease is defined by the ability of tumor cells of the primary site toinvade local tissues and to cross tissue barriers. . . . To initiate themetastatic process, carcinoma cells must first penetrate the epithelialbasement membrane and then invade the interstitial stroma. . . . Fordistant metastases, intravasation requires tumor cell invasion of thesubendothelial basement membrane that must also be negotiated duringtumor cell extravasation. . . . The development of malignancy is alsoassociated with tumor-induced angiogenesis (which) not only allows forexpansion of the primary tumor, but also permits easy access to thevascular compartment due to defects in the basement membranes of newlyformed vessels.”

MIMETIC: a molecule which, in shape and effect, mimics the shape andtherefore the activity of another molecule or complex of molecules uponwhich it is designed.

MUTEIN: An amino acid sequence variant of a protein. The variation inprimary structure may include deletions, additions and substitutions.The substitutions may be conservative or non-conservative. Thedifferences between the natural protein and the mutein generallyconserve desired properties, mitigate or eliminate undesired propertiesand add desired or new properties. In the present invention the muteinsgenerally are those that maintain or increase anti-metastatic activity.Particularly, uteroglobin muteins are amino acid sequence variants ofuteroglobin that maintain or increase the anti-metastatic activity ofuteroglobin.

NSAID: Nonsteroidal anti-inflammatory agents. Small molecule drugs, asthe term is used herein, that inhibit cyclooxygenase, but do notdirectly inhibit phospholipase A₂. These compounds have been used fortheir anti-inflammatory action. Aspirin, phenylbutazone, ibuprofen,sulfinypyrazone (Anturane) and indomethacin are NSAIDs.

PEPTIDE ANALOG: an oligo or polypeptide having an amino acid sequence ofor related to a protein. Peptide analogs of the present invention arepeptides that have anti-metastatic activity and an amino acid sequencethe same as or similar to a region of an anti-metastatic protein, suchas uteroglobin.

PIN: prostatic intraepithelial neoplasia. A condition associated withprostate cancer having two grades, high grade PIN which may indicate ahigh risk of prostate cancer, and low grade PIN which may indicate a lowrisk or prostate cancer. PIN is also known as dysplasia, intraductaldysplasia, large acinar atypical hyperplasia, atypical primaryhyperplasia, hyperplasia with malignant change, marked atypia, andduct-acinar dysplasia. Pin may be characterized by a highnuclear/cytoplasmic ratio, hyperchromasia, coarsely granular chromatin,absence of nucleoli, isolated cells and cellular and nuclearpleomorphism.

PLA: phospholipase A

PLA₂: phospholipase A₂

PREVENTION: in relation to metastasis, virtually complete inhibition, nometastasis if it had not occurred, no further metastasis if there hadalready been metastasis of a cancer. See INHIBITION.

PSA: Prostate specific antigen

RBM: reconstituted basement membrane. A multicomponent matrixapproximating the molecular composition of the intracellular tissuematrix and the epithelial cell basement membrane. Preparations forpreparing RBM are well known and are available from commercialsuppliers.

SFM: Serum free medium

UG: uteroglobin

hUG: human uteroglobin

DETAILED DESCRIPTION OF THE INVENTION

Notwithstanding past failures to develop methods to friendly prostaticintraepithelial neoplasia or to distinguish non-metastatic aberrationsand tumors with low metastatic potential from aberrations and tumorswith high metastatic potential, the present invention provides methodsfor determining the metastatic potential of aberrant growths, tumors andcancers and for identifying prostatic intraepithelial neoplasia.

In addition, notwithstanding past failures to develop effectiveanti-metastatic treatments the present invention provides compounds andcompositions for inhibiting cancer metastases and methods foradministering the compounds and compositions to inhibit or preventmetastasis of a tumor of epithelial cell origin in an organism.

DETERMINING THE METASTATIC POTENTIAL OF TUMORS

Determining the activity of factors that effect arachidonic acid releasein cells can serve to indicate metastatic potential of a tumor. In thisregard, determining PLA₂ activity, and the activity or abundance offactors that affect the activity of PLA₂ can serve to indicatemetastatic potential. Determining metastatic potential in accordancewith this aspect of the present invention is illustrated by thefollowing discussion of uteroglobin protein, mRNA or DNA as an index ofmetastatic potential of prostatic tumors, a very particularly preferredembodiment of the invention, which should not be construed as beinglimitative.

METHODS

Uteroglobin to index metastatic potential

The prostate is the sex gland in males that makes seminal fluid. It islocated, in the standing male, vertically below the bladder, where itsurrounds the urethra. Generally, it is shaped roughly like a slightlyelongated sphere, like a walnut. In most men, it is about an inch indiameter until about age 50 and thereafter it tends to grow larger.

Pathology of the prostate can present as infection, benign enlargementor cancer. Benign growth adversely affects health only when the enlargedprostate constricts the urethra and interferes with urination. Malignantgrowth always poses a threat to health and life of the patient;although, as discussed below, prognoses and indicated treatments varygreatly between occurrences.

Prostate cancer is the most frequently diagnosed cancer in men in theUnited States. Prostate cancers generally do not grow quickly. Usually,they double in size only every three or four years. Adverse affects ofprostate cancer also develop slowly, as they are effected by the growthof the tumor itself.

The slow progression of prostate cancer presents something of aconundrum in men 50 or older, who present the majority of prostatecancer cases. Often the normal progression of prostate cancer suggeststhat debilitating effects will no develop within the normal lifeexpectancy of the patient. Given the lack of effective treatments andthe deleterious side effects attendant to the treatments currentlyavailable waiting may be the best therapy for many elderly patients.

Unfortunately, it is difficult to distinguish benign from canceroustumors and, more importantly, slow growing localized tumors from thoseformed by more aggressive, metastatic cancers. Thus, even the bestphysician cannot accurately predict the course of progression of a givenprostate cancer, and cannot prescribe the best treatment regimen.

In one aspect the present invention overcomes this obstacle to effectivetreatment of such tumors by providing a method to determine themetastatic potential of prostatic tumors. In accordance with this aspectof the invention, uteroglobin protein, mRNA or DNA is determined incells of biopsy material. The protein, mRNA or DNA determined in thecells, by comparison to uteroglobin determined in normal cells,indicates the metastatic potential of prostatic tumors, particularlythose of epithelial cell origin.

It is worth noting in this respect that previous studies did notidentify the relationship between metastatic potential of a tumor anddecreased expression of uteroglobin (or any other inhibitors ofarachidonic acid release). In previous studies, for instance,uteroglobin (called Clara cell 10 kDa protein, abbreviated CC10) wasused as a marker for certain types of cells, and cell-type specificityof its expression was studied. (As described in Linnoila et al.,A.J.C.P. 97(2): 235-243 (1992) and Peri et al., J. Clin. Invest. 92:2099-2109 (1993), which are incorporated by reference herein in theirentirety). In addition, CC10 expression was reported to vary betweenpatients and cell types. In particular, it was reported that CC10expression was lower in lung cancer patients and in smokers without lungcancer than it was in non-smokers, and decreased CC10 expression hasbeen loosely associated with neoplasm. (Broers et al., Lab. Invest. 66:337-346 (1992) and Jensen et al., Int. J. Cancer 58: 629-637 (1994),which are incorporated by reference herein in their entirety. However,no studies of CC10 expression have suggested that uteroglobin expressionin cells of a tumor can be used to determine metastatic potential.

Specific detection of proteins

Proteins indicative of metastatic potential of tumors can be determinedin cells in biopsy material by conventional methods well known to thoseof skill in the art. Such methods are described in many standardtextbooks and laboratory manuals. For instance, the techniques formaking and using antibody and other immunological reagents and fordetecting particular proteins in samples using such reagents aredescribed in CURRENT PROTOCOLS IN IMMUNOLOGY, Coligan et al., Eds., JohnWiley & Sons, New York (1995), which is incorporated by reference hereinin parts pertinent to making and using reagents useful for determiningspecific proteins in samples. As another example, immunohistochemicalmethods for determining proteins in cells in tissues are described inVolume 2, Chapter 14 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubelet al., Eds., John Wiley & Sons, Inc. (1994), which is incorporated byreference herein in part pertinent to carrying out such determinations.Finally, Linnoila et al., A.J.C.P. 97(2): 235-243 (1992) and Peri etal., J. Clin. Invest. 92: 2099-2109 (1992), incorporated herein asreferred to above, describe techniques that may need, in part, in thisaspect of the present invention.

For instance, uteroglobin can be determined in sample in accordance withthe invention by histochemical methods set out in Miyamoto et al., J.Urology 149: 1015-1019 (1993), which is incorporated by reference hereinin its entirety. As described therein, for instance, suitable biopsymaterial is obtained from a patient suspected of having benign prostatichyperplasma or prostatic carcinoma and immediately placed into 0.01Mphosphate buffered saline. Thereafter, the material is immediatelyprocessed. It is mounted on a brass plate using rate liver homogenate asan adhesive. The material then is frozen in liquid nitrogen-cooledisopentane. Sections suitable for assay of uteroglobin in cells of thematerial are sectioned in a cryostat. Sections are obtained across thebiopsy material, avoiding parts of the biopsy material that are damagedor deleteriously altered by the removal process.

Sections are dried at room temperature, fixed and then washed.Paraformaldehyde is a particularly useful fixative in this regard, butmany other fixatives also can be used. The sections may be pretreatedwith hydrogen peroxide and a non-ionic detergent, such as Triton X-100.Also, sections may be incubated with a blocking solution to reducenon-specific binding. For instance, the sections may be incubated withgoat blocking serum prior to incubation with a goat serum, goat antibodyor goat antibody-derived reagent.

Uteroglobin then is visualized for determination in the samples using auteroglobin-specific binding reagent, such as a monoclonal or apolyclonal anti-uteroglobin antibody. Binding of theuteroglobin-specific reagent to cells in the sections may be determineddirectly, if the reagent has been conjugated to a detectable label, orusing a second or additional reagents, such as a secondaryantibody-enzyme conjugate.

In preferred embodiments of the invention, the uteroglobin-specificreagent is an antiserum, a polyclonal antibody, a derivative of apolyclonal antibody, a monoclonal antibody, a derivative of a monoclonalantibody or an engineered antibody, such as a single chain antibody.Derivatives of monoclonal and polyclonal antibodies include conjugatesand fragments. Antibodies conjugated to detectable labels are preferredin this regard. Among detectable labels are enzymes such as horseradishperoxidase. Among fragments preferred in this regard are Fab fragments,F(ab)₂ fragments and F(ab′) fragments.

Sections are incubated with uteroglobin-specific reagent underconditions effective for the uteroglobin-specific reagent to bindefficiently to uteroglobin in said cells, while binding to othercellular components is inefficient; i.e., under conditions effective forthe ratio of specific to non-specific binding to provide accuratedetermination of uteroglobin content in cells of the biopsy material.

At the same time, control sections may be incubated under the sameconditions with a corresponding reagent that is not specific foruteroglobin, to estimate background binding. For polyclonal immuneserum, for instance, control sections can be incubated with preimmuneserum to monitor background, non-specific binding. After the incubationperiod, the specific reagent, and any reagent used in the controls, isremoved, as by washing.

If the primary, uteroglobin-specific reagent is detectably labelled,then the label may be determined and, thereby the uteroglobin content ofcells in the sample. In this case, controls preferably would be labeledand would be determined in like fashion. More often, and preferably, asecondary reagent is used to visualize binding of reagents on thesections, as described below.

After removing unbound specific and non-specific reagents, test andcontrol sections are incubated with a secondary reagent that bindsspecifically to the primary, uteroglobin specific reagent and itscounterpart in the controls. Preferably, the secondary reagent is abiotinylated anti-antibody.

The sections are incubated with the secondary reagent under conditionsfor the reagent to bind efficiently to the primary reagent (and itscounterpart in the controls) in the cells, while binding to othercellular components is inefficient; i.e., under conditions effective forthe ratio of specific to non-specific binding to provide accuratedetermination of uteroglobin content in cells of the biopsy material.

Thereafter, the unbound fraction of the secondary reagent is removedfrom the sections. The secondary reagent, and its counterpart in thecontrols, then is determined. If the secondary reagent comprises adetectable label, incubation with a tertiary reagent generally will notbe necessary. However, use of a tertiary reagent comprising a detectablelabel is more commonly employed for immunocytochemical analysis,generally. Therefore, for illustrative purposes, the three componentassay is described here.

The sections then are incubated with a tertiary reagent comprising adetectable label that binds specifically to the secondary reagent.Incubation is carried out under conditions effective for the tertiaryreagent to bind efficiently to the secondary reagent bound to primaryreagent in cells in the test sections or its counterpart in controlsections, while binding to other cellular components is inefficient;i.e., under conditions effective for the ratio of specific tonon-specific binding to provide accurate determination of uteroglobincontent in cells of the biopsy material. A preferred tertiary reagentcomprising a detectable label is an avidinated enzyme for binding tobiotinylated secondary reagent. A preferred enzyme in this regard ishorseradish peroxidase.

Unbound tertiary reagent is removed, by washing the sections withbuffer, for instance. The detectable label bound in cells in the biopsymaterial then may be determined. In preferred embodiments of theinvention, sections are incubated under conditions effective for anenzyme in the tertiary reagent to catalyze a chromogenic reaction.Binding of the uteroglobin-specific reagent is determined by the colorgenerated by the reaction.

Suitable reagents and conditions for carrying out the determination ofuteroglobin in cells in biopsy samples are well known and readilyavailable. A multiplicity of procedures and reagents can be effectivelyemployed for this purpose. Such reagents and techniques routinely areemployed by those of skill in the arts of immunocytochemistry,histopathology and cytology.

Kits for performing such assays, in whole and in part, are widelyavailable from numerous commercial suppliers. Incubation with secondaryantibodies, and subsequent visualization of uteroglobin, can carried outaccording the given procedures prescribed by commercial suppliers.

In preferred embodiments the sections are stained with hematoxylin andeosin to confirm pathology and to facilitate comparison of uteroglobinin normal and diseased cells in the same section.

In particularly, preferred embodiments, the relative staining ofdiseased and normal cells in a sections is compared with staining infiduciary cells. The fiduciary cells are reference standards whichtypify results obtained by a given procedure in normal cells, cellscharacteristic of benign tumors, and cells characteristic of malignanttumors. Within any category, moreover, fiduciary cells may provide agrated series of characteristic results. Uteroglobin in fiduciary cellsmay be determined at the same time uteroglobin is determined in cells ofthe biopsy sample, or at another time. In a particularly preferredembodiment of the invention, uteroglobin is determined in fiduciarycells which serve as a standard reference series for subsequent clinicalassays.

In normal tissue immunocytochemical technique, such as those describedabove, reveal very heavy staining of uteroglobin in the luminal surfaceof prostatic epithelial cells. Biopsy samples that evidence intermediarypathology thought to precede neoplasia, such as prostaticintraepithelial neoplasia (“PIN”), show a pattern of uteroglobinstaining similar but weaker than that of normal cells. Biopsy materialfrom malignant tumors shows significant decreases in staining ofuteroglobin in cells. The decrease in staining of the luminal surface ofepithelial cells in prostatic tumors is particularly dramatic. Whereas,in normal tissue the luminal surface of epithelial cells shows thehighest staining for uteroglobin, uteroglobin staining either cannot bedetected or is faint in the same cells in metastatic prostatic tissue.

It is this ability to differentiate between normal tissue, prostaticintraepithelial neoplasia, and cancer which provides for theidentification of PIN. Due to the association of PIN with cancer,especially high-grade PIN which has been reported to have a 70%association with cancer, this provides an early diagnosis, prognosis andtreatment of prostate cancer.

Furthermore, it has been reported that there is a close relationshipbetween the age-related prevalence of PIN and the age-related prevalenceof prostate cancer with the occurrence of PIN mirroring the occurrenceof prostate cancer but having a 20-30 year lag time. It is thisrelationship which provides a practitioner the diagnostic and prognosticability of early detection and treatment.

The risk of developing invasive cancer is gauged by the decrease inuteroglobin in diseased cells in the biopsy sample relative to cells innormal tissue of the same type, as described above. In biopsy materialcontaining both normal and diseased tissue the staining of cells in thenormal and diseased tissue can be compared on the same section. Ingeneral, the cells in low grade relatively confined tumors expressuteroglobin in amounts similar to normal cells. The cells in aggressive,invasive tumors express little or no uteroglobin and are poorlydifferentiated in their morphology.

Specific detection of mRNA and DNA

mRNA also can be determined in cells in biopsy samples to determinemetastatic potential. mRNA can be determined by a variety of methodswill known to those of skill in the art, which can be carried out usingwell known and readily available starting materials, including thosewidely available from commercial suppliers. Techniques useful in thisregard are described in the foregoing references. Techniques that may beparticularly pertinent in this regard relating to uteroglobin aredescribed in Broers et al., Lab. Invest. 66:337-346 (1992) and Jensen etal., Int. J. Cancer 58:629-637 (1994), incorporated herein as referredto above.

A given mRNA may determined in cells of biopsy tissue by in situhybridization to a specific probe. Such probes may be cloned DNAs orfragments thereof, RNA, typically made by in vitro transcription, oroligonucleotide probes, usually made by solid phase synthesis. Methodsfor making and using probe suitable for specific hybridization in situare ubiquitously known and used in the art.

By specific hybridization is meant that the probe forms a duplex withthe given, target mRNA that is stable to the conditions of hybridizationand subsequent incubations and that duplexes formed between the probeand other, non-target mRNAs are not stable and generally do not persistthrough subsequent incubations. Specific hybridization thus means thatthe ratio of hybridization to target and non-target mRNAs provides anaccurate determination of the target mRNA in cells in the biopsy sample.

In a particularly preferred embodiment of the present invention a probethat hybridizes specifically to uteroglobin mRNA is used to determineuteroglobin mRNA in cells of biopsy tissue, particularly prostaticbiopsy material.

Techniques suitable for in situ determination of target mRNAs, such asuteroglobin mRNA, are described in a variety of well known and readilyavailable laboratory manuals, as well as the primary literature. Anillustrative procedure from the primary literature in this regard isdescribed in Broers et al. Laboratory Investigation 66 (3):337-346(1992), which is incorporated by reference herein in its entirety.

In general, biopsy material is obtained by suitable surgical procedureand snap frozen, as by freezing in methybutane/dry ice. The samples canbe embedded and sectioned much as described above for the determinationof protein is biopsy samples. Sections can be thawed onto and affixed toglass slides previously cleaned with acid and ethanol and coated withpoly-L-lysine. The tissue sections thereafter can be exposed to bufferedformaldehyde, acetylated, treated with buffered glycine and thenprehybridized in 50% formamide, 2 X SSC (where 1 X is 0.15M NaCl, 0.015Msodium citrate, pH 7.0). After prehybridization the sections can behybridized to the labelled probe in 50% formamide, 10% dextran sulfate,2 X SSC.

The exact conditions of the steps in the procedure, especially theprehybridization, hybridization and criterion steps will be adjustedwith the T_(m) (or the T_(d)) of the probe and to provide the desireddegree of specificity of hybridization; i.e., the desired stringency.

Theoretical approximations and empirical methods for determining properconditions in this regard are well known and routinely practiced bythose skilled in the pertinent arts. Approximation calculations andexperimental techniques in this regard are described, for instance, inSambrook et al. (1989) referred to herein above.

Those of skill will appreciate, for instance, that the formamide in theforegoing solutions serves to provide equivalent hybridizationconditions at lower temperature. For instance, hybridization in 50%formamide at about 50° C. provides conditions similar to hybridizationat 65° C. without formamide. The lower temperature of hybridization canhelp preserve the biopsy sections during the hybridization procedure,aiding subsequent identification and examination of cells and mRNAcontent. Other agents that preserve features of the tissue sections thataid analysis likewise are preferred.

Dextran sulfate generally is used to accelerate the hybridizationreaction and to drive it to completion in a shorter period of time, asis well known. Similar agents that increase the rate of hybridization,consistent with accurate determination of specific mRNA content, alsoare useful in the present invention.

Following hybridization, the probe-containing solution and unbound probeare removed. Typically, the sections are washed several times withprehybridization buffer, such as 50% formamide, 2 X SSC, at or slightlyabove the hybridization temperature.

If an RNA probe is used for detection of the target mRNA, the sectionsthen are treated with RNAseA, typically in the same solution, and thenwashed to remove RNAseA and byproducts with 50% formamide, 2 X SSC underthe same conditions as the previous washings.

Finally, the sections typically are washed several additional times in 2X SSC at room temperature and then air dried.

Radioactive probes generally are visualized by autoradiography. For thispurpose slides can be dipped in a photographic emulsion, dried andallowed to expose the emulsion at 4° C. for an appropriate period oftime. Using a preferred emulsion, NTB-2 nuclear track emulsion, exposuretimes of 3 to 7 days are appropriate. The exposure time can be alteredby a variety of factors including the use of more highly labelledprobes.

The emulsions are developed at the end of the exposure period and then,typically, counterstained with hematoxylin and eosin. Subsequently,labelling of target mRNA in cells can be assessed by microscopy usingbrightfield and darkfield illumination.

As discussed above regarding protein, the abundance and distribution ofthe target mRNA in cells in the biopsy section indicates the metastaticpotential of the tumor. Particularly, the relative abundance of mRNA indiseased and normal cells indicates metastatic potential.

A variety of controls may be usefully employed to improve accuracy inassays of this type. For instance, sections may be hybridized to anirrelevant probe and sections may be treated with RNAseA prior tohybridization, to assess spurious hybridization.

Thus, for instance, as discussed for uteroglobin protein, normal tissueexhibits high concentrations of uteroglobin mRNA in the prostaticepithelial cells. Intermediary pathology, such as prostaticintraepithelial neoplasia (“PIN”), shows a lower concentration ofuteroglobin mRNA similar to but less than the hybridization to auteroglobin mRNA-specific probe exhibited by normal cells. Biopsymaterial from malignant tumors shows significant decreases in theconcentration of mRNA uteroglobin in cells by exhibiting little or nohybridization to a uteroglobin mRNA-specific hybridization probe.

Again, the differentiation between normal tissue, prostaticintraepithelial neoplasia, and cancer provides for the identification ofPIN and an early diagnosis, prognosis and treatment of prostate cancer.

aberrant mRNA or DNA

Some metastatic tissues of prostatic origin exhibit seemingly normalhybridization to a uteroglobin-specific probe, even though cells in thesame tissue do not synthesize much, if any, uteroglobin protein. Thesecells typically exhibit aberrant uteroglobin mRNA, rather decreaseduteroglobin mRNA. For instance, aberrant splicing has been demonstratedin at least one human prostatic carcinoma.

Splicing and other aberrations in mRNAs of cells of metastatic tissuecan be determined by northern and southern blotting techniques and byPCR techniques. These techniques also are well known to those of skillin the art and can be applied readily to the determination of mRNA incells of biopsy material in accordance with the present invention.

Techniques employed to assess restriction fragment length polymorphisms(“RFLP”) can be applied to detect some mutations associated withaberrant splicing patterns. The assessment always can be made on themRNA, but in some dysfunctions it can be made on the genomic DNA aswell. The mRNA or DNA can be amplified prior to RFLP analysis, as well,using PCR or other suitable technique.

In addition to RFLP techniques, SSCP can be used to detect aberrantsplicing of messages, such as uteroglobin-specific mRNA. For thispurpose, a target mRNA, such as uteroglobin mRNA, is amplified byreverse transcriptase-mediated PCR. The double-stranded amplified DNA isdenatured and run on gels in which mobility is quite sensitive to smallchanges in secondary structure.

Yet another technique that can be employed to determine aberrantsplicing, among other things, is ligase mediated PCR. This techniquealso is well known to those of skill in the art, and techniques suitableto the analysis and determination of mRNAs and genomic aberrations thathave been described in the literature readily can be applied to thedetermination of aberrant mRNAs in cells of tumor.

In regard to all of the foregoing, the determination of mRNA and genomicDNA in epithelial cells in biopsy material is preferred. Particularlypreferred in this regard is prostatic biopsy material.

Among probes and hybridization targets for determination of metastaticpotential of tumors by determination of target mRNA and DNA are probesspecific for uteroglobin mRNA or for aberrations of uteroglobin mRNA oruteroglobin-encoding DNA indicative of altered expression of uteroglobinand, therefore, of metastatic potential.

FIDUCIARIES

Fiduciaries may be developed in accordance with this aspect of theinvention, to guide interpretation of results. In this regard, a proteinor mRNA in accordance with the foregoing may be determined in biopsymaterial from representative tumors of a specific type, characteristicof a specific degree of metastatic potential.

Characterization in this regard may benefit from hindsight, followingthe actual course of tumor progression in patients as they undergotreatment and thereafter. Fiduciary results characterizing a gradedseries of metastatic potential also may obtained from cell culturestudies, as described elsewhere herein, illuminated in the examplesbelow.

The determination of a protein or mRNA, or other agent, as set outabove, in a variety of tumors of known metastatic characteristic, andthe correlation of the determinations with metastatic potential isanother preferred embodiment of the present invention.

KITS

Reagents for carrying out the methods described above may beincorporated into kits for use in determining the metastatic potentialof a tumor or identifying prostatic intraepithelial neoplasia. All ofthe techniques and reagents discussed herein with regard to thedetermination of metastatic potential and identifying prostaticintraepithelial neoplasia, including reagents and methods set out in theexamples below may be included in these kits. Preferred kit componentsgenerally are those that detect the agents discussed elsewhere herein,particularly as discussed in the foregoing sections pertaining todetermination of a protein or mRNA diagnostic of metastatic potential orPIN.

The kits also may include one or more fiduciary results, such asreference slides of immunocytochemical results characteristic of atumors with high and low metastatic potential, or reference slices of insitu hybridization results characteristic in the same regard. Preferablyin this regard, are kits that include a fiduciary series forinterpreting results. The fiduciary may be in the form of one or morephotographs or may be depicted in other ways, including writtendescriptions. In addition, the fiduciary may be highlytumor-type-specific or it may be applicable to related types of tumors.

ANTI-METASTATIC AGENTS AND METHODS

The invention disclosed herein provides agents and methods forinhibiting or preventing metastasis. The following discussionillustrates the invention in this respect.

Anti-metastatic agents

In particular, in accordance with this aspect of the invention, whichinclude compounds that inhibit arachidonic acid release by cells of atumor of epithelial cell origin in an organism are administered by aroute and in an amount effective to prevent or inhibit metastasis of thetumor.

Inhibitors of arachidonic acid release

Without being limited to any theory of the invention, applicants notethat arachidonic acid is a substrate in the synthetic pathway ofeicosanoids in cells. Various eicosanoids play a role in stimulating orinhibiting shape, attachment, motility and proliferation of cells. Insome aspects of the invention, inhibiting arachidonic acid release incells of tumors of epithelial cell origin inhibits or extinguishesmetastatic potential.

Inhibitors of phospholipase A₂

Compounds that inhibit phospholipase A₂ (PLA₂) are preferred compoundsof the present invention. PLA₂ is a membrane signaling enzyme of thearachidonic acid cascade, the series of enzymes, substrates, productsand co-factors involved in the production and secretion of arachidonicacid, and it generally will be the case that inhibitors of PLA₂ activitygenerally will inhibit release of arachidonic acid. Notably, PLA₂ hasbeen associated with processes of inflammation, rather thantumorigenesis or metastasis, and it has been suggested as a target forthe control of chronic inflammation, but not as a target for developingan anti-metastatic agent. Nevertheless, the present invention providescompositions and methods of PLA₂ inhibitors for inhibiting metastasis oftumors of epithelial cell origin. The formulation and use of thesecompounds in the invention is illustrated by reference to the preferredembodiments discussed below.

Especially preferred among PLA₂ inhibitors are lipocortins, muteins oflipocortins, peptide analogs of lipocortins and uteroglobins, muteins ofuteroglobins and peptide analogs of uteroglobin. Uteroglobins, muteinsof uteroglobins and peptide analogs of uteroglobin are particularlypreferred. Most particularly preferred are uteroglobins, and among thesehuman uteroglobin is very especially preferred. The discussion below,directed to uteroglobin, particularly human uteroglobin, illustrates theinvention is this regard.

Uteroglobins

Uteroglobin, also called blastokinin, was first discovered as a majorprotein component of the rabbit uterine fluid during early pregnancy.The human counterpart to rabbit uteroglobin was first found innonciliated Clara cells in the distal bronchiole airway and wasoriginally designated Clara cell 10 kD protein, abbreviated as “CC10.”Uteroglobin also has been detected in humans in the uterus, respiratorytract, and prostate gland, by immunohistochemical methods.

The complementary DNA for human uteroglobin (CC10) has been closed andits sequence has been determined, as reported in Singh et al., BBA950:329-337 (1988), incorporated by reference herein in its entirety.

Uteroglobin has been purified to homogeneity by at least two groups andit has been structurally and functionally characterized in considerabledetail. In brief, uteroglobin occurs in rabbits as a dimer of twoidentical chains. The monomers are 70 amino acids long. They arearranged antiparallel to one another in the dimer. Also, in the dimerthey are covalently linked by two symmetrical disulfide bonds, formedbetween ‘Cys-3’ and “Cys-69” and, reciprocally, between ‘Cys-69’ and“Cys-3” (where ′ designates one chain in the dimer and ″ designates theother chain). Each monomer chain contains four α-helical segments and aβ-turn, the later at Lys-26 to Gln-29. The structure, function andactivities of uteroglobin has been reviewed, for instance, by Miele etal., Endocrine Reviews 8:474-490 (1987), which is incorporated byreference herein in its entirety.

Uteroglobin inhibits the activity of PLA₂, as shown by in vitro assays.Generally, it has been thought to have immunomodulatory oranti-inflammatory activities, or both, that act to protect the wetepithelia of organs that communicate with the external environment.Uteroglobin expression is steroid-sensitive and its secretion in theendometrium has been shown to be stimulated by progesterone. Uteroglobinalso has been reported to have an anti-chemotactic effect on neutrophilsand monocytes. Uteroglobin has not been seen as playing a role in canceror metastasis. Thus, it was surprising to find that uteroglobin, inaccordance with the present invention, can be used to inhibit or preventmetastasis of a tumor of epithelial cell origin in an organism.

Without being bound to any theory of the mechanism by which uteroglobininhibits metastasis, it appears, as the Examples show, that theinhibitory action of uteroglobin on metastasis results from inhibitionof PLA₂ activity and inhibition of arachidonic acid release by the tumorcells. Any uteroglobin may be useful in the invention that inhibitsarachidonic acid release that inhibits or prevents metastasis of a tumorof epithelial cell origin. Uteroglobins for use in the invention may berecovered from natural sources, it may be made by recombinant means, itmay be produced by chemical techniques, it may be made by semi-syntheticmethods or it may be obtained by a combination of techniques.

Methods for purifying uteroglobin to homogeneity from a natural sourcehave been described in Nieto et al., Arch. Biochem. Biophys. 180:80-92(1977), which is herein incorporated by reference in its entirety. Othermethods for this purpose can be equally useful in this regard.

The most highly preferred uteroglobin for use in the invention at thepresent time is human uteroglobin. Preferably, human uteroglobin for usein the invention is made by expression of a cloned gene in a host cellin cultures or in an animal. Techniques for expressing uteroglobin inthis way are well known to those of skill in the art.

A cDNA encoding human uteroglobin, useful toward this end, has beenisolated, sequenced and expressed in cells in culture. Methods forexpressing cloned DNAs that encode uteroglobin have been describedspecifically with regard to human uteroglobin in Mantile et al., J. BiolChem. 268:20343-20351 (1993) and Miele et al., J. Biol. Chem.265:6527-6435 (1990), which, as noted below, are incorporated byreference herein in their entirety.

Techniques for obtaining, manipulating and expressing cloned genes toobtain uteroglobin for use in the present invention are well known tothose of skill in the art and are described in protocol-like detail in avariety of laboratory manuals. For instance, such methods are set forthin Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2ND Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989),the entirety of which is herein incorporated by reference.

Lipocortins

Lipocortin proteins also are known as annexins, and are well known tothe art. They generally have been characterized as calcium-dependentphospholipid-binding proteins. For instance, Arcone et al., Eur. J.Biochem, 211:347-355 (1993), incorporated by reference herein in itsentirety, reports on the structure of human lipocortin 1 and theexpression of the active protein using an expression vector in E. coli.

Lipocortins have been implicated in the mechanism of anti-inflammatoryactivity of glucocorticosteroids. Furthermore, anti-inflammatoryactivity has been associated with the amino-terminal end of humanlipocortin 1, as shown by the activity of acylated polypeptidecorresponding to human lipoprotein residues 2-26, synthesized andcharacterized by Cirino et al., J. Pharmacol. 108:573-4 (1993), which isincorporated by reference herein in its entirety.

According to one hypothesis, lipocortins mediate aglucocorticosteroid-dependent inhibition of phospholipase A₂. Somestudies support a “substrate-depletion” mechanism of phospholipase A₂inhibition, as reported by Bastian et al., J. Invest. Dermatol.101:359-63 (1993), for instance, which is incorporated by referenceherein in its entirety.

The studies have focused on the intermediary role of lipocortins in theanti-inflammatory response to glucocorticosteroids, however, and a rolefor the lipocortin proteins in metastasis was unknown.

In accordance with one aspect of the present invention, lipocortins maybe used to inhibit or present metastasis of tumors of epithelial cellorigin. The methods of using lipocortins, and the compositions oflipocortins, in accordance with the present invention, will beunderstood by reference to the discussion elsewhere herein, particularlyby reference to the illustrative disclosure relating to uteroglobin andto prostate cancers.

Lipocortins generally may be used in accordance with this aspect of thepresent invention. Preferred lipocortins have a high therapeutic effectand low incidence of deleterious side effects. Particularly preferredlipocortins are those of human origin. Human lipocortin 1 is among thoseparticularly preferred.

Muteins and peptide analogs

Techniques such as those described in the foregoing manual can be usedto make variants and analogs of uteroglobin and other proteins useful inthe invention. Recombinant DNA methods, chemical synthetic methods,enzymatic methods and mixed methods for making, altering and utilizingmuteins and peptide analogs are well known and are described here onlybriefly to illustrate their applicability to the present invention.

-muteins

It will be appreciated by those of skill that cloned genes readily canbe manipulated to alter the amino acid sequence of a protein. The clonedgene for human uteroglobin can be manipulated by a variety of well knowntechniques for in vitro mutagenesis, among others, to produce variantsof the naturally occurring human protein, herein referred to as muteins,that may be used in accordance with the invention.

The variation in primary structure of muteins of lipocortins oruteroglobins useful in the invention, for instance, may includedeletions, additions and substitutions. The substitutions may beconservative or non-conservative. The differences between the naturalprotein and the mutein generally conserve desired properties, mitigateor eliminate undesired properties and add desired or new properties. Inthe present invention the muteins generally are those that maintain orincrease anti-metastatic activity. Particularly, uteroglobin muteins areamino acid sequence variants of uteroglobin that maintain or increasethe anti-metastatic activity of uteroglobin.

-peptide analogs

Similarly, techniques for making small oligopeptides and polypeptidesthat exhibit activity of larger proteins from which they are derived (inprimary sequence) are well known and have become routine in the art.Thus, peptide analogs of proteins of the invention, such as peptideanalogs of lipocortin and uteroglobin that exhibit anti-metastaticactivity also are useful in the invention.

Mimetics

Mimetics also can be used in accordance with the present invention toprevent or inhibit metastasis of tumors. The design of memetics is knownto those skilled in the art, and generally are understood to be peptidesor other relatively small molecules that have an activity the same orsimilar to that of a larger molecule, often a protein, on which they aremodeled.

Thus, by way of illustration, uteroglobin mimetics, for instance, can beused in accordance with the present invention in the same manner asuteroglobin itself, to prevent or inhibit metastasis of a tumor.

The design of such mimetics can be based on the structure-functionrelationship of uteroglobin. By studying the effect of mutations onanti-metastatic activity of uteroglobin the sites in the proteinresponsible for anti-metastatic activity can be identified. In vitromutagenesis procedures that can be used to systematically alter clonedgenes, such as cDNAs encoding uteroglobin and other proteins withanti-metastatic activity of the present invention, are described inSambrook et al. (1988). Systematically mutagenized proteins, alsoreferred to as muteins as noted elsewhere herein, can be produced usingsuch altered DNA by standard methods for expression cloned genes inorganisms to produce heterologous proteins. Such methods are well knownto those of skill and are described in, for instance, Sambrook et al.(1988) referred to herein above. The muteins so produced then can beassayed for anti-metastatic activity, using in vitro or in vivo assaysthat model or measure metastatic activity. Suitable methods aredescribed herein and illustrated in the examples below.

Methods for determining aspects of protein structure also are well knownto those of skill in the art. To some extent, the structure of a givenprotein can be approximated by analogy to structures of relatedproteins. Physical and chemical information about a protein structurecan be obtained by a wide variety of well known techniques, includingactive site modification techniques, NMR, and X-ray crystallography.

This information can be combined with information from studies thatcorrelate structural alterations with changes in activity, such as themutagenesis studies described above, to generate a map of the shape andchemical functions important to a given activity of a protein.

Molecules that mimic the shape and chemical functionality that providethe desired activity, then can be designed and synthesized. Computermodeling methods that can be employed toward this end, as well asmethods of organic synthesis, peptide synthesis and for the synthesis ofother classes of compounds that can be used to produce mimetics inaccordance with this aspect of the invention are well known to those ofskill in the art.

Once a mimetic has been designed and synthesized, it can be assayed foranti-metastatic activity using techniques for this purpose, such asthose described elsewhere herein.

Results of activity studies and of structural studies of the mimeticsthemselves can be used to design further mimetics that are moreeffective, have fewer undesirable side effects, or have additionalactivities, such as by combining two mimetics in a single molecule.

In the same manner as for uteroglobin, mimetics can be designed forother compounds that have anti-metastatic activity. Preferred in thisregard, as described above, as anti-metastatic mimetic compounds thatinhibit arachidonic acid release by cells of a cancer of epithelial cellorigin. Particularly preferred are mimetics that inhibit phospholipaseA₂ in such cells. In this regard, mimetics of uteroglobin or lipocortinare especially preferred. Among the most highly preferred mimetics inthis regard are mimetics of human uteroglobin.

Small molecule drugs

Compounds other than the proteins, muteins, protein-derived peptides,mimetics and the like discussed above, that inhibit arachidonic acidrelease by cells of cancers of epithelial cell origin also may be usefulin the present invention. Among such compounds are certain small organicmolecules, which may be mimetics, that inhibit arachidonic acid release.Inhibition may be mediated by inhibition of PLA₂ activity, or byinhibition of other enzymes or intermediates involved in metabolicinteractions that result in arachidonic acid release.

Among such compounds is the anti-inflammatory agent mepacrine, which hasbeen shown to inhibit PLA₂, and an experimental drug, indomethacin,which has been shown to inhibit cyclooxygenase. Both compounds andexhibit anti-metastatic activity in in vitro assays, at doses that havebeen shown to be non-toxic in patients. Both compounds, thus, can beutilized in accordance with the present invention.

PLA₂, as noted herein above, is a key enzyme in the arachidonic acidcascade. As noted above, inhibitors of PLA₂ are most preferred in theinvention, in this regard. Among small molecule drugs, mepacrine ispreferred among inhibitors of PLA₂. Other relatively small moleculedrugs (small, in this case, meaning small relative to proteins ofaverage size) that, like mepacrine, inhibit PLA₂ also will be useful inthe invention, in the same fashion as mepacrine and the other PLA₂inhibitors discussed herein above. Among small molecule drugs, such PLA₂inhibitors are particularly preferred. In this regard, mepacrine ishighly preferred and other compounds that are similar to mepacrine inchemical structure are particularly preferred.

Cyclooxygenase is the key enzyme in the cyclooxygenase-dependent pathwayof arachidonic acid metabolism, wherein arachidonic acid is a precursorin the synthesis of prostaglandins, prostacyclins and thromboxanes.Among small molecule drugs, inhibitors of cyclooxygenase also arepreferred for use in the invention disclosed herein. Nonsteroidalanti-inflammatory agents (“NSAIDs”) are among the small molecule drugs,as the term is used herein, that inhibit cyclooxygenase and arepreferred in the invention in this regard. NSAIDs are described, forinstance, in PRINCIPLES OF PHARMACOLOGY, Munson et al., EDs., Chapman &Hall, New York (1995), which is incorporated herein by reference in partpertinent thereto, including, particularly, Chapter 74.

Among NSAIDs in accordance with this aspect of the invention areaspirin, phenylbutazone, ibuprofen, sulfinpyrazone (Anturane) andindomethacin. In this regard, indomethacin is particularly preferred andcompounds that are similar to indomethacin in chemical structure alsoare preferred.

Lipoxygenase is the key enzyme in the lipoxygenase-dependent pathway ofarachidonic acid metabolism, wherein arachidonic acid is a precursor inthe synthesis of thromboxanes. Inhibitors of lipoxygenase, and otherdownstream enzymes of the lipoxygenase-dependent pathway also may be ofuse in the present invention.

Compositions

Any non-toxic, inert and effective carrier may be used to formulatecompositions of the present invention. Well known carriers used toformulate other therapeutic compounds for administration to humansparticularly will be useful in the compositions of the presentinvention. Pharmaceutically acceptable carriers, excipients and diluentsin this regard are well known to those of skill, such as those describedin the MERCK INDEX, 11th Ed., Budavari et al., Eds., Merck & Co., Inc.,Rahway, N.J. (1989), which is incorporated by reference herein in itsentirety. Examples of such useful pharmaceutically acceptableexcipients, carriers and diluents include distilled water, physiologicalsaline, Ringer's solution, dextrose solution, Hank's solution and DMSO,which are among those preferred for use in the present invention.

In particular, for instance Mantile et al., J. Biol Chem.268:20343-20351 (1993), incorporated by reference hereinabove, report onsterile formulated, lyophilized uteroglobin that may be useful inpreparing uteroglobin compositions of the invention.

Cancers

Methods and compositions of the present invention may be applied to thetreatment of a variety of cancers of epithelial cell origin. Among theseare metastatic cancers of breast, lung, colon, bladder, prostate,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges and the skin. An especially preferredtarget is prostate cancer, particularly prostate cancer of epithelialcell origin.

The following detailed discussion of prostate cancers is provided inillustration of the compositions and methods of the invention not onlyas to prostate cancers, but also other cancers that may be treated inanalogous or identical fashion, in accordance with the presentinvention.

Prostatic adenocarcinoma

Adenocarcinoma of the prostate is one of the most common malignancies.It is estimated that 240,000 new cases of prostate cancer will bediagnosed in the United States in 1995, and that it will cause more than50,000 deaths during one year. In fact, prostate adenocarcinoma is thesecond leading cause of cancer-related mortality in the United States.

With prostate cancer, as with all solid tumors, it is the metastaticencroachment of the tumor on other vital function that causes the demiseof the patient. Approximately 10% of patients are diagnosed initiallywith metastatic disease. Ultimately, 30-40% of patients with this cancerwill develop metastatic disease. Once metastasis occurs there is a thecancer follows a relentless progression.

Invasion is a prerequisite for migration of tumor cells. In connectivetissue, stroma and basement membranes form the major physical barriersto the migration process. Invasion of the local extracellular matrix(ECM) by tumor cells thus can be marked as the first step in metastasis.The sequential biochemical mechanism of ECM invasion first involves cellattachment to specific components of ECM followed by a progressivecascade of proteolytic dissolution. Prostate cancers which grow to acritical size exhibit extracapsular invasion and metastasize to specificanatomical sites apparently in response to stromal cell secretoryproteins which are necessary for their growth and proliferation.Invasive migration of tumor cells within the prostate gland may occur asa function of chemokinesis along anatomical paths of least resistance,such as the perineural duct. Further establishment of metastasis reliesupon successful penetration of the circulatory or lymphatic system,followed by vessel extravasation at the secondary organ, whichfrequently is bone tissue for cancers of prostatic origin. Nearly all ofthese steps, including attachment, matrix degradation and migration, canbe modeled experimentally in vitro by measuring invasion of areconstituted basement membrane (RBM) barrier in response tofibroblast-conditioned medium (FCM) which serves as a chemo-attractant.

In vivo, of growth and proliferation of prostate tumor cells primarilyis responsive to stromal cell (fibroblast) secretory proteins.Extracapsular invasiveness of prostate tumor cell can be modeled bymigration of tumor cells in vitro into reconstituted basement membrane(RBM) in the presence and absence of a chemoattractant, such asfibroblast conditioned medium (FCM). The assay determines cells thathave attached to the RBM, degraded the RBM enzymatically and, finally,cells that have towards the FCM side of the membrane. The events in thein vitro invasion assay comport with the important steps observed in themetastatic spread of tumor cells through the basement membrane in vivo.

Prostate tumors frequently initially metastasize to regional lymphnodes, having disseminated through the lymphatic circulation. They alsospread to other sites through the vascular system, which is extensivelyinterconnected to the lymphatics. The final site of formation ofmetastasis is a function of a number of parameters, including: (i) thefirst capillary bed encountered by blood vessels draining the tumor, and(ii) organ preference of the tumor cells with respect to characteristicsof specific tissues that nurture attachment and growth of tumor cellswith metastatic potential.

Metastatic potential of prostate cancers of epithelial cells origin canbe inhibited by compositions and methods of the invention. Inparticular, metastasis of these cancers can be inhibited by humanuteroglobin, as shown by the examples set out herein below.

Route of administration

Therapeutic treatment with uteroglobin can utilize any type ofadministration including topical, other non-invasive and invasive means.

Administration by non-invasive means may be by oral, intranasal ortransdermal routes, among others.

Generally, at the present time, invasive techniques are preferred.Administration by invasive techniques may be intravenous,intraperitoneal, intramuscular or directly in tumors, among others.

Administration may be by a single dose, it may be repeated at intervalsor it may be continuous. Since uteroglobin is small, easily diffusible,and relatively stable it is well suited to long-term continuousadministration, such as by perfusion pump. Where continuousadministration is applied, infusion is preferred. In this situation,pump means often will be particularly preferred for administration.Especially, subcutaneous pump means often will be preferred in thisregard.

In other situations it will be desirable to administered uteroglobin andother agents of the present invention by intramuscular self-injection ona regular basis.

Compositions and methods of the invention also may utilize controlledrelease technology. Thus, for example, uteroglobin may be incorporatedinto a hydrophobic polymer matrix for controlled release over a periodof days. Such controlled release films are well known to the art.Examples of polymers commonly employed for this purpose that may be usedin the present invention include nondegradable ethylene-vinyl acetatecopolymer and degradable lactic acid-glycolic acid copolymers. Certainhydrogels such as poly(hydroxyethylmethacrylate) or poly (vinylalcohol)also may be useful, but for shorter release cycles then the otherpolymer releases systems, such as those mentioned above.

Dose

The quantity of the active agent for effective therapy will depend upona variety of factors, including the type of cancer, means ofadministration, physiological state of the patient, other mendicantsadministered, and other factors.

Treatment dosages generally may be titrated to optimize safety andefficacy. Typically, dosage-effect relationships from in vitro studiesinitially will provide useful guidance on the proper doses for patientadministration. Studies in animal models also generally may be used forguidance regarding effective dosages for treatment of metastatic cancersin accordance with the present invention.

These considerations, as well as effective formulations andadministration procedures are well known in the art and are described instandard textbooks, such as GOODMAN AND GILMAN'S: THE PHARMACOLOGICALBASES OF THERAPEUTICS, 8th Ed., Gilman et al. Eds. Pergamon Press (1990)and REMINGTON'S PHARMACEUTICAL SCIENCES, 17th Ed., Mack Publishing Co.,Easton, Pa. (1990), both of which are incorporated by reference hereinin their entirety.

Typical therapeutic doses will be about 0.1 to 1.0 mg/kg of body weightof pure uteroglobin. The does may be adjusted to attain, initially, ablood level of about 0.1 μM.

A particular formulation of the invention uses a lyophilized form ofuteroglobin, in accordance with well known techniques. For instance, 1to 100 mg of uteroglobin may be lyophilized in individual vials,together with carrier and buffer compounds, for instance, such mannitoland sodium phosphate. The uteroglobin may be reconstituted in the vialswith bacteriostatic water and then administered, as described elsewhereherein.

Administration regimen

Any effective treatment regimen can be utilized and repeated asnecessary to affect treatment.

In clinical practice, the compositions containing uteroglobin orrecombinant uteroglobin, alone or in combination with other therapeuticagents are administered in specific cycles until a response is obtained.

For patients who initially present without metastatic disease,uteroglobin-based drugs can be used as an immediate initial therapyprior to surgery and radiation therapy, and as a continuouspost-treatment therapy in patients at risk for recurrence or metastasis(based upon high PSA, high Gleason's score, locally extensive disease,and/or pathological evidence of tumor invasion in the surgicalspecimen). Therapy for these patients aims, for instance, to decreasethe escape of potentially metastatic cells from the primary tumor duringsurgery or radiotherapy, decrease the escape of tumor cells fromundetectable residual primary tumor, decrease tumor cell attachment tothe interior vessel wall, decrease the migration of tumor cells out ofthe vessel, and thereby decrease invasion into the interstitial spacesof the distal organ.

For patients who initially present with metastatic disease,uteroglobin-based drugs can be used as a continuous supplement to, orpossible as a replacement for hormonal ablation. A goal of therapy forthese patients is to slow tumor cell escape from both the untreatedprimary tumor and from the existing metastatic lesions in order to slowthe progressive encroachment of further metastases.

In addition, the invention maybe particularly efficacious duringpost-surgical recovery, where the present compositions and methods maybe particularly effective in lessening the chances of recurrence of atumor engendered by shed cells that cannot be removed by surgicalintervention.

Gene therapy

Certain embodiments of the present invention relate to anti-metastaticgene therapy. Gene therapy is a new approach to treatment of diseases.Currently, gene therapy protocols relate to therapy of certain carefullychosen disorders, including certain inherited disorders, a number ofaggressively fatal cancers and AIDS. The restricted application of genetherapy to a few disorders reflects concerns about the efficacy, safetyand ethical implications of the approach in general, and currenttechniques in particular. Despite the cautious approach mandated bythese concerns, and despite the fact that techniques for carrying outgene therapy are still in an early stage of development, results fromthe first few trials have been very encouraging, some spectacularly so.It seems certain that gene therapy techniques will improve rapidly andthat gene therapies soon will develop into an increasingly important andubiquitous modality for treating disease. (Reviewed, for instance, inTolstoshev, Ann. Rev. Pharm. Toxicol. 32: 573-596 (1993) and Morgan etal., Ann. Rev. Biochem. 62:191-217 (1993), which are incorporated byreference herein in their entirety).

The delivery of a variety of therapeutic agents clearly will beaccomplished by gene therapy techniques. Many of the procedures now inuse or under current development for gene therapy may be used inaccordance with the present invention to prevent or inhibit metastasis.Additional techniques that will be developed in the future similarlywill be found useful in the present invention. The following discussionis illustrative of the use of gene therapy techniques to prevent orinhibit metastasis in accordance with the present invention.

By gene therapy, in the following discussion, generally is meant the useof a polynucleotide, in a cell, to achieve the production of an agentand the delivery of the agent to a tumor in situ, i.e., in a patient, toengender an anti-metastatic effect. The agent may itself be aanti-metastatic agent or it may engender the production of ananti-metastatic agent upon introduction into the patient.

Approaches to genetic therapy currently being developed, which can beused in accordance with this aspect of the invention disclosed herein,often are grouped into two major categories: ex vivo and in vivotechniques.

Ex vivo techniques generally proceed by removing cells from a patient orfrom a donor, introducing a polynucleotide into the cells, usuallyselecting and growing out, to the extent possible, cells that haveincorporated, and, most often, can express the polynucleotide, and thenintroducing the selected cells into the patient. Cells that target tumorcells in vivo, including tumor cells that have migrated from primary orsecondary tumor sites, generally are preferred in this type of genetherapy.

In addition, as described further below, the polynucleotide may beintroduced directly into the patient. The polynucleotide in this casemay be introduced systemically or by injection into a tumor site. Thepolynucleotide may be in the form of DNA or RNA, alone or in a complex,or in a vector, as discussed further below.

The polynucleotide may be in any of a variety of well-known forms, forinstance, a DNA, a DNA fragment cloned in a DNA vector, a DNA fragmentcloned in DNA vector and encapsidated in a viral capsid.

The polynucleotide may be an RNA or a DNA. More typically it is a DNA.It may include a promoter, enhancer and other cis-acting control regionsthat provide a desired level and specificity of expression in the cellsof a region operably linked thereto that encodes an RNA, such as ananti-sense RNA, or a protein. The polynucleotides may contain severalsuch operably linked control and encoding regions for expression of oneor more mRNAs or proteins, or a mixture of the two.

Preferred in this regard are polynucleotides that encode theanti-metastatic agents described herein above. As noted in the foregoingdiscussion, inhibitors of arachidonic acid release are preferred.Inhibitors of PLA₂ activity are particularly preferred. Among PLA₂inhibitors, uteroglobins and lipocortins are particularly preferred,uteroglobins especially, human uteroglobin particularly amonguteroglobins.

Muteins and polypeptide analogs of protein inhibitors also are useful inthe invention and may be encoded by polynucleotides for gene therapy toinhibit metastasis. In this regard, muteins and the polypeptide analogsof the foregoing preferred embodiments also are preferred in this aspectof the invention.

In addition, peptide mimetics that can be encoded by a polypeptide forsynthesis in cells can be used in accordance with this aspect of theinvention. Preferred embodiments in this regard those set out above.

The polynucleotide may be introduced into cells either ex vivo or invivo, including into the tumor in situ. A variety of techniques havebeen designed to deliver polynucleotides into cells for constitutive orinducible expression, and these routine techniques can be used in genetherapy of the present invention as well. Polynucleotides will bedelivered into cells ex vivo using cationic lipids, liposomes or viralvectors. Polynucleotides will be introduced into cells in vivo,including into cells of tumors in situ, using direct or systemicinjection. Methods for introducing polynucleotides in this manner caninvolve direct injection of a polynucleotide, which then generally willbe in a composition with a cationic lipid or other compound or compoundsthat facilitate direct uptake of DNA by cells in vivo. Such compositionsmay also comprise ingredients that modulate physiological persistence.In addition, polynucleotides can be introduced into cells in vivo inviral vectors.

Genetic therapies in accordance with the present invention may involve atransient (temporary) presence of the gene therapy polynucleotide in thepatient or the permanent introduction of a polynucleotide into thepatient. In the latter regard, gene therapy may be used to repair adysfunctional gene to prevent or inhibit metastasis.

Genetic therapies, like the direct administration of agents discussedabove, in accordance with the present invention may be used alone or inconjunction with other therapeutic modalities.

Combined with other treatments

Uteroglobin may be used in conjunction with other treatment modalities.Other common treatment modalities are discussed below specifically byreference to prostate cancer. It will be appreciated that similarconsideration will apply to treatment of other metastatic cancers. Thepresent invention may be used in conjunction with any current or futuretherapy.

Surgery and Radiation

In general, surgery and radiation therapy are employed as potentiallycurative therapies for patients under 70 years of age who present withclinically localized disease and are expected to live at least 10 years.Neither treatment modality has a significant role in the management ofmetastatic diseases, and neither treatment is generally performed ifmetastasis is present at initial diagnosis.

Approximately 70% of newly diagnosed prostate cancer patients fall intothis category. Approximately 90% of these patients (63% of totalpatients) undergo surgery, while approximately 10% of these patients (7%of total patients) undergo radiation therapy.

Histopathological examination of surgical specimens reveals thatapproximately 63% patients undergoing surgery (40% of total patients)have locally extensive tumors or regional (lymph node) metastasis thatwas undetected at initial diagnosis. These patients are at asignificantly greater risk of recurrence or metastasis. Approximately40% of these patients will actually develop recurrence or metastasiswithin 5 years after surgery. Results after treatment with radiation areeven less encouraging. Approximately 80% of patients who have undergoneradiation as their primary therapy have disease persistence or developrecurrence or metastasis within 5 years after treatment.

Currently, surgical and radiotherapy patients generally do not receiveany immediate follow-up therapy. Rather, they typically are monitoredfor elevated Prostate Specific Antigen (“PSA”), which is the primaryindicator of recurrence or metastasis.

Thus, there is considerable opportunity to use the present invention inconjunction with surgical intervention.

Hormonal Therapy

Hormonal ablation is the most effective palliative treatment for the 10%of patients presenting with metastatic disease at initial diagnosis.Hormonal ablation by medication and/or orchiectomy is used to blockhormones that support the further growth and metastasis of prostatecancer. With time, both the primary and metastatic tumors of virtuallyall of these patients become hormone-independent and resistant totherapy. Approximately 50% of patients presenting with metastaticdisease die within 3 years after initial diagnosis, and 75% of suchpatients die within 5 years after diagnosis.

In this regard, it may be worth noting that the natural uteroglobin geneis dependent upon hormones for expression, and hormonal ablation maydecrease expression of the endogenous uteroglobin gene, both in tumorcells and in the normal tissue surrounding the tumor. Auteroglobin-deficient state could render the patients more susceptibleto successful metastasis. Continuous supplementation withuteroglobin-based drugs may be used to prevent or reverse thispotentially metastasis-permissive state from developing in hormonaltherapy treatment modalities.

Chemotherapy

Chemotherapy has been more successful with some cancers than withothers. It is likely that the combination of chemotherapy with therapiesof the present invention in some cases will be synergistic. Chemotherapycurrently has little effect on prostate cancer and is used only as alaser resort, with universally dismal results.

Immunotherapy

The present invention also can be used in conjunction withimmunotherapies. Not only may the methods and compositions hereindisclosed be used with the increasing variety of immunological reagentsnow being tested or used to treat cancer, but it also may be used withthose that come into practice in the future. The present invention thusmay be used with immunotherapies based on polyclonal or monoclonalantibody-derived reagents, for instance. Monoclonal antibody-basedreagents are among those most highly preferred in this regard. Suchreagents are well known and are described in, for instance, RitterMONOCLONAL ANTIBODIES—PRODUCTION, ENGINEERING AND CLINICAL APPLICATIONS,Ritter et al., Eds., Cambridge University Press, Cambridge, UK (1995),which is incorporated by reference herein in its entirety. Radiolabelledmonoclonal antibodies for cancer therapy, in particular, also are wellknown and are described in, for instance, CANCER THERAPY WITHRADIOLABELLED ANTIBODIES, D. M. Goldenberg, Ed., CRC Press, Boca Raton,Fla. (1995), which is incorporated by reference herein in its entirety.

Cryotherapy

Cryotherapy recently has been applied to the treatment of some cancers.Methods and compositions of the present invention also can be used inconjunction with an effective therapy of this type.

Compositions comprising several active agents

According to another aspect of the invention, pharmaceuticalcompositions of matter useful for inhibiting cancer metastases areprovided that contain, in addition to the aforementioned compounds, anadditional therapeutic agent. Such agents may be chemotherapeuticagents, ablation or other therapeutic hormones, antineoplastic agents,monoclonal antibodies useful against cancers and angiogenesisinhibitors. The following discussion highlights some agents in thisrespect, which are illustrative, not limitative. A wide variety of othereffective agents also may be used.

Among hormones which may be used in combination with the presentinvention diethylstilbestrol (DES), leuprolide, flutamide, cyproteroneacetate, ketoconazole and amino glutethimide are preferred.

Among antineoplastic and anticancer agents that may be used incombination with the invention 5-fluorouracil, vinblastine sulfate,estramustine phosphate, suramin and strontium-89 are preferred.

Among the monoclonal antibodies that may be used in combination with theinvention CYT356 is preferred.

The present invention is further described by reference to thefollowing, illustrative examples.

EXAMPLE 1 Preparation of Human Uteroglobin by Gene Expression

Human uteroglobin was purified from E. coli cells expressing afull-length cDNA. The methods for obtaining the cDNA, constructing itinto a vector for expression in host, expressing the construct andpurifying the protein all involve art routine techniques. Such methodsare described specifically, with regard to human uteroglobin in Singh etal., BBA 950: 329-337 (1988), Mantile et al., J. Biol Chem. 268:20343-20351 (1993) and Miele et al., J. Biol. Chem. 265: 6427-6435(1990), which are incorporated by reference herein in their entirety.

Briefly, in the present illustrative example, which followed thetechniques set out in the foregoing references, a clone containing afull-length cDNA encoding human uteroglobin in the well known vectorpGEM4Z was digested with PstI. (pGEM4Z may be obtained from Promega,Inc. Many other equally suitable vectors also are availablecommercially.) The digestion freed a 340-base pair fragment containingall of the cDNA and 53 nucleotides of the pGEM4Z polylinker. Thefragment was purified by preparative gel electrophoresis in low meltingtemperature agarose. The purified fragment was ligated into the Pstlsite of the expression vector pLD101, downstream of an induciblepromoter. The ligation and subsequent cloning produced the plasmidpGEL101. This construct was introduced into E. coli strain BL21 (DE3)cells for expression of uteroglobin protein.

For expression, bacteria were cultured under routine conditions for E.coli growth, and then induced for uteroglobin expression by making themedia 0.45 mM in IPTG (isopropyl-1-thio-D-galactopyranoside). Afterappropriate further incubation to accumulate expressed protein, thecells were collected and then lysed. Uteroglobin was purified from thelysed cells using standard methods of size exclusive and ion exchangechromatography.

EXAMPLE 2 Cells for Assays of Metastatic Potential

The cell lines used in the illustrative embodiments herein discussed arewell known and readily available. The four cell lines of the presentexamples all were divided from human prostate cancer and are ofepithelial cell origin. TSU-Pr1, DU-145 and PC3-M areandrogen-independent. LNCaP is androgen-sensitive.

DU-145 is described in Stone et al., Int. J. Cancer 21: 274-281 (1978),which is incorporated herein by reference in its entirety. The cell lineis available from a variety of sources including, for instance, theAmerican Type Culture Collection (Rockville, Md.).

LNCaP is described in Horoszewicz et al., Cancer Res. 43: 1809-1818(1983), which is herein incorporated by reference in its entirety. Thiscell line may be obtained from, for instance, the American Type CultureCollection (Rockville, Md.).

PC3-M is described in Kaighn et al., Invent. Urol. 11:16-23 (1976) whichis herein incorporated by reference in its entirety.

TSU-Pr1 is described in Hzumi et al., J. Urology 137:1304-1306 (1987)which is incorporated by reference in its entirety.

Cells of each line were grown and maintained in monolayer culture inαMEM (minimal essential medium) supplemented with glutamine, 10% fetalbovine serum, penicillin (100 units/ml) and streptomycin (100 (g/ml).Cultures were incubated at 37° C. in 5% CO₂/95% air. Media was replacedevery second day.

EXAMPLE 3 General Assay for Invasiveness of Cells

1. Culture

As described briefly below, invasiveness of cells was assayed by themethods described in Albini et al., Cancer Research 47: 3239-3245(1987), which is incorporated herein by reference in its entirety.Invasiveness assays and other methods for assessing anti-metastaticaffects, as discussed herein below are described in Leyton et al.,Cancer Research 54:3696-3699 (1994) which is incorporated by referenceherein in its entirety.

Cells in logarithmic phase were detached from the growth surface bybrief exposure to 0.25% trypsin, 0.25% EDTA, collected and centrifugedat 800× g for 5 min. The pellet was resuspended in SF medium, countedand seeded into 6 mm dishes, 1.5×10⁶ cells per dish. The cells then wereincubated for 24 hr in media containing zero, 0.01, 0.1 and 1.0 μMuteroglobin. After the incubation cells were gently collected using arubber policeman and assayed for invasiveness.

Fibroblast conditioned media (FCM) served as a chemo-attractant tostimulate invasion. It was prepared by culturing proliferating 3T3 cellsfor 24 hr. in SF medium and then collecting the media, free of cells.The cell free media thus obtained served as FCM.

Invasiveness was measured using a polycarbonate membrane precoated witha reconstituted basement membrane. Well known RBMs are suitable for thispurpose. For example, Albini et al., supra describes REM of the typeemployed for these experiments.

Assays were performed in blind-well Boyden chambers. The lowercompartment of each chamber was filled with 220 μl of FCM, aschemo-attractant, or 220 μl serum-free media, as control for basalinvasiveness. A polycarbonate membrane (12 μm pore size), coated with 25μg/50 μl RMB, was placed over the lower compartment. Tumor cells forassay were added to the upper compartment, 3.0×10⁵ cells per well, andthe chambers then were incubated at 37° C. for 6 hr.

(Reconstituted basement membrane preparations for use in accordance withthe foregoing assay are readily available from numerous commercialsuppliers. One suitable example membrane in this regard is “MATRIGEL”sold by Collaborative Biomedical Products of Bedford, Mass.)

2. Quantitation

Invasive activity was measured by the number of cells that penetratedthe RBM, as determined by a technique involving crystal violet stainingdeveloped for use with the Boyden chamber. The technique, summarizedbelow, is well known and is described, for instance, in Frandson et al.,Fibrinolysis 6(Supp4): 71-76 (1992), which is incorporated by referenceherein in its entirety.

The RMB-coated membrane was removed from each chamber at the end of theincubation period. The filters were pinned down to a wax plate, keepingthe surface with the invading cells upward. The cells were stained onthe filters with 0.5% crystal violet in 25% methanol for 10 minutes.Then, the filters were rinsed in distilled water, four times or untilcrystal violet no longer leached into the wash water. After the wash,the surface of each filter that had been in contact with the wax platewas carefully wiped clean with a moist cotton swab, to removenonmigrating cells. The filters then were placed in a 24-well clusterplate and dried overnight.

Crystal violet in the invading cells on each filter was extracted twicefor 10 minutes into 500 μl aliquots of 0.1 M sodium citrate, 50%ethanol. The amount of crystal violet in the extract was analyzed byabsorbance at 585 nm, using a standard spectrometer.

3. Analyses

Assays were carried out in triplicate for each data point.

Variance between control and test groups was analyzed for significanceusing the standard repeated measures test for analysis of variance.P<0.01 generally was considered indicative of a significant effect, withexceptions, as noted elsewhere herein.

EXAMPLE 4 Correlation of Optical Density with Cell Counts, and Assay ofBasal Invasiveness

To calibrate optical density of the crystal violet extracts against thenumber of migrating cells on filters, cells were counted, seeded atknown densities on filters, incubated to allow attachment and washed.One set of a duplicate set of plates was used for cell counting. Theother set was used for staining. For counting, cells were released fromthe filters by mild trypsinization and then counted using an automatedcell counter. For staining, after washing, the cells were stained andcrystal violet stain then was extracted from the stained cells, asdescribed in EXAMPLE 3. The optical densities of the extracts weremeasured by standard spectroscopy. The optical density determined foreach filter extract was matched with the number of cells attached to itscompanion filter, as determined by direct counting. These paired datapoints served to correlate optical density with cell counts. plottedthat displayed the

In accordance with the foregoing procedure, several densities of DU-145cells were seeded into the top chamber of the Boyden jars. The jars wereset up in pairs, and for each pair dye uptake by the cells was measuredon one filter and the number of cells was counted on the other filter,as described above.

The results are shown in Table I, which sets out the number of cellsmigrating to the lower face of the filters as a function of the numberof cells seeded in the upper chamber. The number of cells migrating tothe lower filter surface also is set out as a percentage of the totalnumber of cells seeded in the top chamber. The data in the Table are themeans of triplicate determinations for each condition, and the indicatedvariance is the standard error of the mean.

At cell seedings greater than 2×10⁵ approximately 22% of cells invadedthe RBM and migrated through the filter in 6 hr. Seeding higher numbersof cells did not increase invasiveness at 6 hr. (Not shown.)

The cells were counted using an automated cell counter, such as the“COULTER MULTISIZER” made by Coulter, Inc. of Hialeah, Fla. By theseexperiments it was determined that an absorbance of 0.1 units of thecrystal violet extract corresponds to approximately 5000 cells thatmigrated through the filter.

Similar procedures can be applied to calibrate the migration assay forother cells, to employ the assay to measure the therapeutic activity ofother compositions of the present invention.

TABLE 1 Relationship between cell invasion and optical absorbance CellsCells seeded O.D. units^(a) invading Percentage (×10³) (585 nm) (×10¹)invasion 100 0.60 ± 0.1 31.8 ± 0.2 31.8 ± 0.2 200 1.50 ± 0.2 42.0 ± 2.121.2 ± 1.0 300 1.20 ± 0.2 72.3 ± 2.7 23.0 ± 0.4 ^(a)1 O.D. unitcorresponds to approximately 5,000 cells.

As shown in FIG. 1, cells of the TSU-Pr1 cell line exhibited the highestrate of basal invasiveness and the highest FCM-stimulated invasiveness.The basal rate for DU-145 cells was 2-fold lower than the rate forTSU-Pr1 cells, but the rate of stimulated invasiveness was comparablefor the two cell lines. As shown in FIG. 2, basal and stimulatedinvasiveness of PCM-3 cells both were 2-fold lower than that observedfor DU-145 cells. (N. B. The Scale change in FIG. 2.) LNCaP cellsexhibited the lowest basal and the lowest stimulated invasiveness. FCMstimulation increased the invasiveness of this cell line 2-3-fold, alsoas shown in FIG. 2.

EXAMPLE 5 Uteroglobin Inhibits Invasiveness of Tumor Cells

The ability of uteroglobin to inhibit tumor cell invasiveness isillustrated by its effect on cell lines treated with 0.01, 0.1 or 1.0 μMuteroglobin. Each of the four cells lines was incubated for 24 hr. withthese concentrations of uteroglobin. After the incubation period thecells were rinsed and then assayed for invasiveness. Inhibition ofstimulated invasiveness then was quantitatively determined bysubtracting the basal invasiveness from FCM-stimulated invasiveness, foreach treatment group. Finally, the results were calculated as apercentage of the invasiveness of untreated control cells.

All four cell lines showed a dose-dependent inhibition of FCM-stimulatedinvasion, as shown in FIGS. 1 and 2, by the bars for FCM/UG. Notably,uteroglobin did not affect basal invasiveness, indicated by the barslabelled SFM/UG. Table 2 shows the average inhibition observed in threeindependent experiments, each of which was performed in triplicate. Theinhibition of invasiveness by uteroglobin was found to be significant asthe P<0.01 level for all conditions, except for PC3-M and TSU-Pr1 cellstreated with 0.01 μM uteroglobin, which were significant at the P=0.05level. As shown in Table 2, 1.0 μM uteroglobin inhibited invasiveness ofDU-145 cells by 60%, PC3-M cells by 88%, LNCaP cells by 92% and theTSU-Pr1 cells by 59%.

TABLE 2 Inhibition of tumor cell invasiveness by uteroglobin %Inhibition of invasion by uteroglobin Cell lines 0.01 μM 0.1 μm 1.0 μMDU-145 45.4 ± 6^(a ) 49.9 ± 5^(a ) 60.2 ± 11^(a) PC3-M 43.4 ± 15^(b)82.4 ± 14^(a) 87.9 ± 11^(a) TSU-Pr1 33.5 ± 10^(b) 44.4 ± 11^(a) 58.9 ±8^(a ) LNCaP 71.5 ± 10^(a) 81.3 ± 6^(a ) 92.3 ± 7^(a ) ^(a)P ≦ 0.001;i.e., statistically significant at the 0.001 level. ^(b)P ≦ 0.05; i.e.,statistically significant at the 0.05 level.

The table shows that results of assays described herein above. Briefly,tumor cells were cultured in media containing uteroglobin for 24 hr. andthen assayed for invasive activity. Basal migration was subtracted andthe adjusted measure is expressed as percent of untreated control cellsfor each cell type. Data is expressed as mean of three independentexperiments performed in triplicate, and the variance is the standarderror of the mean.

EXAMPLE 6 Time Course of Inhibition of Invasiveness by Uteroglobin

The time course for suppression of invasiveness of DU-145 cells byuteroglobin was determined over a 24 hr. period. Cells were treated, asdescribed above, for 3, 6, 12, or 24 hr. and then assayed forinvasiveness. The maximum inhibition of invasiveness observed in thecells cultured in the presence of uteroglobin was 74%, at 12 hr. 50%maximum inhibition was observed after 3 hr., and at 24 hr. inhibitionwas 79% of the maximum. FIG. 4 shows these results in graphical form.

EXAMPLE 7 Uteroglobin Does Not Affect Simple Motility

Experiments carried out to assess motility per se show that uteroglobindoes not affect normal cell motility, i.e., migration in the absence ofRMB. The results show that uteroglobin specifically inhibits theinvasion-associated motility of epithelial tumor cells.Invasion-associated motility, which is implicated more specifically inmetastasis than motility per se, involves the synthesis, recruitment, oractivation of several different classes of proteolytic enzymes includingcollagenases, cathepsins, plasminogen activators and a variety ofmetalloproteinases required for degradation of basement membranes andthe ECM. The observation that uteroglobin does not alter cell motility,but inhibits FCM-stimulated invasiveness, indicates that uteroglobin,specifically can inhibit metastatic invasiveness without directlyaltering motility of normal cells. This is an advantageous property forpharmacological intervention where non-specific effects of uteroglobinon normal motility could be disadvantageous.

EXAMPLE 8 Uteroglobin Does Not Affect Adhesion to RBM

The anti-invasive activity of uteroglobin is not mediated by an effecton cell adhesion. This can be seen from experiments in which theadhesiveness of DU-145 and PC3-M cells was measured after incubation for24 hr. in SM media or SM media containing 1.0 μM uteroglobin for 24 hr.Adhesion was tested by removing the incubation media, resuspending thecells in fresh αMEM/SM, counting them, replating the cells and thencounting the number of cells that had attached 1, 3 and 6 hr. afterplating. The results show that uteroglobin does not alter adhesivenessof the cells. The absence of an effect of uteroglobin on basalmigration, illustrated by the results depicted in FIGS. 1 and 2,supports the same conclusion. This is an advantageous property forpharmacological intervention where non-specific effects of uteroglobinon normal cell adhesiveness could be disadvantageous.

EXAMPLE 9 Specificity of Uteroglobin Anti-metastatic Effects

The specificity of uteroglobin activity was demonstrated in DU-145cells, using myoglobin, albumin and heat inactivated uteroglobin. Cellswere treated for 24 hr with either myoglobin, albumin or uteroglobinthat had been inactivated by incubation at 55° C. for 45 min. Theresults, presented in the graph in FIG. 3, show that myoglobin, albuminand heat-inactivated uteroglobin do not effect invasive activity oftumor cells.

EXAMPLE 10 Uteroglobin Inhibits Arachidonic Acid Release by FCMStimulated Tumor Cells

The effect of uteroglobin on the release of arachidonic acid (“AA”) bytumor cells was assayed under conditions of basal and stimulatedinvasiveness. (¹⁴C)AA having a specificity activity 58.0 mCi/mmol wasused to trace the arachidonic acid release. Labelled arachidonic acid ofthis type can be obtained from several commercial suppliers, such asAmersham, Inc. of Arlington Heights, Ill.

Uteroglobin inhibits release of arachidonic acid by DU-145 cellsstimulated by FCM, as shown by the following experiment.

Intracellular arachidonic acid in DU-145 cells was labeled by incubatingthe cells in media containing ¹⁴C-labelled arachidonic acid. For thispurpose approximately 0.75×10⁵ cells were incubated for 24 hr. at 37° C.in 2 ml of α-MEM/SF media containing 1 μCi of (¹⁴C)AA. After this, thecells were washed three times with 20 ml of 0.2% bovine serum albumin toremove free radioactivity.

The washed, labelled cells were resuspended in 2 ml of α-MEM/SF, FCM orFCM containing 1.0 μM uteroglobin. Cells in each of the three media wereincubated at 37° C. and 50 μl aliquots were removed of the media wereremoved from each culture 0.5, 10, 20 and 30 min., and 1, 2, 3, 4 and 5hr. after the beginning of the incubation period.

Each aliquot was assayed for AA release, which was measured at ¹⁴C freein the media, determined by scintillation counting. Standardscintillents and counters were used to quantitate radiation emission by¹⁴C in the samples, e.g., EcoLite Biodegradable scintillation from ICN,Inc.

Stimulation of AA release by FCM was calculated by subtracting theamount of (¹⁴C)AA released by cells incubated in αMEM/SF media (whichwas very low) from the amount of (¹⁴C)AA released by cells incubated inFCM media. The effect of uteroglobin on FCM-stimulated AA release wascalculated in the same way, by subtracting the amount of (¹⁴C)AAreleased by cells cultured in αMEM/SF from the amount of (¹⁴C)AAreleased by cells incubated in FCM containing 1 μM uteroglobin.

As shown in the graph in FIG. 5, arachidonic acid released by cellscultured in FCM media exhibited a biphasic profile. Released AA peakedat 20 min., the peak was followed by a period of reuptake, e.g. 60 min.and then there was a period of sustained release to the end of the 5 hr.incubation period of this experiment.

The presence of 1 μM uteroglobin in FCM media reduced FCM-stimulatedrelease of arachidonic by 77% at 20 min. and 86% at 5 hr.

The dramatic inhibition of release of arachidonic acid by FCM-stimulatedtumor cells, together with the foregoing results showing the inhibitoryeffect of uteroglobin on invasiveness, show that uteroglobin affects anearly event in the signalling pathway(s) that control tumorinvasiveness.

EXAMPLE 11

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and after surgeryat a dose rate that reaches and then maintains a blood concentration ofuteroglobin of approximately 1 μM. After post-operative recovery, thepatient is maintained at a decreased level of uteroglobin by a regimenof periodic i.m. self-administration. No further occurrences of theadenocarcinoma develop.

EXAMPLE 12

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration by subdural pump. Nofurther occurrences of the adenocarcinoma develop.

EXAMPLE 13

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears not to have metastasized. The adenocarcinoma isremoved by surgery. Uteroglobin is administered before and aftersurgery, at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin of approximately 1 μM. After post-operativerecovery, the patient is maintained at a decreased level of uteroglobinby intermittent or continuous administration using a transdermal patch.No further occurrences of the adenocarcinoma develop.

EXAMPLE 14

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. No furthertumors develop.

EXAMPLE 15

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Althoughsome of the original, small tumorous masses are detected after surgery,they do not grow in size.

EXAMPLE 16

A patient presents with metastatic adenocarcinoma of the prostate. Theadenocarcinoma appears to have metastasized, but surgery still isindicated as an effective treatment modality. Tumor tissue is removed bysurgery. Uteroglobin is administered from the time, approximately, ofthe initial diagnosis and continues after surgery, i.m. and i.v., at adose rate that reaches and then maintains a blood concentration ofuteroglobin above 1 μM. After post-operative recovery, the patient ismaintained at this level of uteroglobin by a regimen of periodic i.m.self-administration. The patient is monitored carefully for intolerableadverse side-effects of high-dose uteroglobin administration. Tumorousmasses are detected after surgery, but their growth is slowed.

EXAMPLE 17

A patient presents with a tumor of the breast, of epithelial cellorigin. The tumor, which appears to be of a metastatic type, appears notto have metastasized. The tumor is removed by surgery. Uteroglobin isadministered after surgery, i.m. and i.v., at a dose rate that reachesand then maintains a blood concentration of uteroglobin of approximately1 μM. After post-operative recovery, the patient is maintained at adecreased level of uteroglobin by a regimen of periodic i.m.self-administration. No further occurrences of the tumor develop.

EXAMPLE 18

A patient presents with a breast tumor of epithelial cell origin. Thebreast tumor has metastasized. Numerous secondary tumors are detected.Insofar as possible, tumor tissue is removed by surgery. Surgicalintervention is aggressive. Uteroglobin is administered from the time,approximately, of the initial diagnosis and continues after surgery,i.m. and i.v., at a dose rate that reaches and then maintains a bloodconcentration of uteroglobin above 1 μM. After post-operative recovery,the patient is maintained at this level of uteroglobin by a regimen ofperiodic i.m. self-administration. The patient is monitored carefullyfor intolerable adverse side-effects of high-dose uteroglobinadministration. No further tumors develop in the remaining breast orelsewhere in the body.

EXAMPLE 19 Uteroglobin is Not Expressed or Secreted by ProstaticAdenocarcinoma Cells

A 76 year old male underwent radical perineal prostatectomy because of abiopsy-based diagnosis of prostatic adenocarcinoma with a Gleason'sscore of 4. Postsurgical definitive pathologic diagnosis showedmoderately to poorly differentiated adenocarcinoma with a Gleason'sscore of 8, nodular prostatic hyperplasia, and multifocal high gradeprostatic intraepithelial neoplasia (PIN). Perineural and lymphaticinvasion was noted, but the seminal vesicles were free of tumor.

With informed consent and in accordance with approved procedures,prostatic tissue was obtained from the diseased prostate gland afterremoval, for evaluation of uteroglobin expression. Tissue fragments werefrozen in liquid nitrogen. Individual slices of frozen tissue weresectioned by cryostat and mounted on silanated microscope slides.

Samples were fixed with 4% formalin for 3 minutes at room temperatureand washed for 10 min in phosphate buffered saline (PBS) pH 8.0.Nonspecific reactivity was blocked by incubating samples with rabbitserum (1:100 dilution) for 30 min. The samples were then exposed to a1:1,000 dilution of goat anti-human uteroglobin primary antibodyovernight at 4 degrees C. Control samples were exposed to a 1:100dilution of goat serum.

All samples were then exposed to biotinylated rabbit anti-goat antibody(1:10,000) for 30 min and then washed with PBS for 10 min. Streptavidincomplex was added for 15 min and the samples washed again with PBS for10 min. DAB was reacted with the samples for 2 min followed by standardstaining of the tissue with hematoxylin-eosin.

Slides were analyzed by two certified pathologists, who were notinformed of the identity of any slides and who carried out theiranalyses independently.

In each case, the normal prostatic tissue stained strongly positive foruteroglobin in epithelial cells, especially at the intracellular luminalsurface. Stromal cells were negative. Areas of hyperplasia diagnosedindependently by both pathologists as PIN stained positively (PIN isconsidered by some to represent an early pre-neoplastic precursor). Incontrast, tumorous epithelial cells exhibited little or no staining foruteroglobin, demonstrating that the tumor cells had lost the ability tosynthesize and secrete this protein.

EXAMPLE 20 Uteroglobin mRNA is not Detected or is Aberrantly Processedin Cells Derived from Metastases of Human Prostatic Tumors

RNA was isolated from normal prostate tissue and from the DU-145, PC-3and TSU-PR1 cell lines derived from metastatic human prostate tumors.The RNA was subjected to Northern blotting according to routine andstandard procedures, using a radiolabelled uteroglobin cDNA probe.Autoradiographic analysis showed that the normal tissue expresses anabundant normally processed 600 base pair mRNA uteroglobin transcript.In contrast the metastatic tumor cells either did not detectably expressthe uteroglobin transcript (DU-145 and PC-3) or expressed a grosslyaberrant transcript (TSU-PR1).

EXAMPLE 21

A patient presents with urinary obstruction and after digital rectalexam a biopsy of the prostate is taken. The initial presurgical reportassigns a Gleason's score of 3 suggesting a low-grade localized tumor. Aportion of the biopsy sample is analyzed immunohistochemically foruteroglobin expression which is found to be present in the normal tissuebut absent in the tumor cells. The diagnosis, prognosis, and plan fortherapy is appropriately altered to reflect the high probability, basedon lack of uteroglobin expression, that the tumor is actually of highergrade than initially diagnosed and probably invasive and metastatic.Uteroglobin therapy is immediately begun.

EXAMPLE 22

After radical prostatectomy, a patient presents with high grademetastatic prostatic adenocarcinoma that has become refractory tohormonal therapy. The patient refused chemotherapy based on its dismalefficacy against prostate cancer and its devastating side effects. Insitu hybridization analysis of a tumor biopsy reveals that theuteroglobin gene is not being expressed in the tumor cells. Furtheranalysis of uteroglobin gene structure by SCCP and RFLP indicate thethat the gene is muted and dysfunctional. The patient chooses to becomea candidate for gene therapy. The patient is injected with anadenovirus-based plasmid expression vector containing the uteroglobingene linked to the promoter of the PSA gene which is specificallyexpressed in prostate cells. The vector is encapsulated in liposomeswhich have anti-PSA antibody fixed on the surface. The antibody-liposomecomplex binds specifically to cells secreting PSA which presumably areonly the metastatic tumor cells. The liposomes are ingested by the cellsand release the plasmids which incorporate into the cells' genomic DNAand begin expressing uteroglobin. The transfected cells expressinguteroglobin reverse their invasive phenotype, thereby ceasing furthermetastasis and are gradually destroyed by the bodies natural defenses.The metastatic tumors regress and the patient's life is prolonged.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention and all suchmodification are intended to be included within the scope of thefollowing claims.

What is claimed is:
 1. A method of inhibiting primary tumor cell growth,comprising administering uteroglobin to a patient in need thereof. 2.The method of claim 1, wherein the administration of uteroglobinprevents the primary tumor cells from damaging surrounding lymph orcirculatory systems.
 3. The method of claim 2, wherein shed cells fromthe primary tumor cells are prevented from entering into the lymph orcirculatory systems.
 4. The method of claim 1, wherein the primary tumorcells are of a tumor of epithelial cell origin.
 5. The method of claim1, wherein the tumor is selected from the group consisting of a breast,lung, colon, bladder, prostate, gastrointestinal track, endometrium,tracheal-bronchial tract, pancreas, liver, uterus, nasopharynges, andskin tumor.
 6. The method of claim 5, wherein the tumor is a prostatetumor.
 7. The method of claim 1, wherein the uteroglobin is humanuteroglobin.
 8. The method of claim 1, wherein the uteroglobin isadministered in combination with another treatment.
 9. The method ofclaim 8, wherein the other treatment is selected from the groupconsisting of surgical intervention, radiation therapy, hormonaltherapy, immunotherapy, chemotherapy, cryotherapy, and gene therapy. 10.The method of claim 1, wherein the uteroglobin is administered directlyto lungs of the patient.
 11. A pharmaceutical composition for inhibitingprimary tumor cell growth, comprising: (i) uteroglobin, and (ii) apharmaceutically acceptable carrier.
 12. The composition of claim 11,wherein the primary tumor cells are of a tumor of epithelial cellorigin.
 13. The composition of claim 11, wherein the uteroglobin ishuman uteroglobin.
 14. The composition of claim 11, wherein thecomposition is administered with an additional therapeutic agent. 15.The composition of claim 14, wherein the additional therapeutic agent isselected from the group consisting of a chemotherapeutic agent, ablationor other therapeutic hormone, antineoplastic agent, monoclonal antibodyuseful against cancer, and angiogenesis inhibitor.
 16. A method ofinterfering with invasion of a local extracellular matrix by tumor cellscomprising administering uteroglobin to a patient need thereof.
 17. Themethod of claim 16, wherein the tumor cells are of a tumor of epithelialcell origin.
 18. The method of claim 17, wherein the tumor is selectedfrom the group consisting of a breast, lung, colon, bladder, prostate,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges, and skin tumor.
 19. The method of claim18, wherein the tumor is a prostate tumor.
 20. The method of claim 16,wherein the uteroglobin is human uteroglobin.
 21. The method of claim16, wherein the uteroglobin is administered in combination with anothertreatment.
 22. The method of claim 21, wherein the other treatment isselected from the group consisting of surgical intervention, radiationtherapy, hormonal therapy, immunotherapy, chemotherapy, cryotherapy, andgene therapy.
 23. The method of claim 16, wherein the uteroglobin isadministered directly to lungs of the patient.
 24. A pharmaceuticalcomposition for interfering with invasion of a local extra-cellularmatrix by tumor cells, comprising: (i) uteroglobin, and (ii) apharmaceutically acceptable carrier.
 25. The composition of claim 24,wherein the tumor cells are of a tumor of epithelial cell origin. 26.The composition of claim 24, wherein the uteroglobin is humanuteroglobin.
 27. The composition of claim 24, wherein the composition isadministered with an additional therapeutic agent.
 28. The compositionof claim 27, wherein the additional therapeutic agent is selected fromthe group consisting of a chemotherapeutic agent, ablation or othertherapeutic hormone, antineoplastic agent, monoclonal antibody usefulagainst cancer, and angiogenesis inhibitor.
 29. A method of inhibitingcell surface receptor interactions with phospholipase A₂ comprisingadministering uteroglobin to a patient in need thereof.
 30. The methodof claim 29, wherein the inhibition prevents invasion of a tumor. 31.The method of claim 30, wherein the tumor is selected from the groupconsisting of a tumor of the breast, lung, colon, bladder, prostate,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges, and skin.
 32. The method of claim 31,wherein the tumor is a prostate tumor.
 33. The method of claim 29,wherein the uteroglobin is human uteroglobin.
 34. The method of claim29, wherein the uteroglobin is administered in combination with anothertreatment.
 35. The method of claim 34, wherein the other treatment isselected from the group consisting of surgical intervention, radiationtherapy, hormonal therapy, immunotherapy, chemotherapy, cryotherapy, andgene therapy.
 36. The method of claim 29, wherein the uteroglobin isadministered directly to lungs of the patient.
 37. A pharmaceuticalcomposition for inhibiting cell surface receptor interactions withphospholipase A₂, comprising: (i) uteroglobin, and (ii) apharmaceutically acceptable carrier.
 38. The composition of claim 37,wherein the uteroglobin is human uteroglobin.
 39. The composition ofclaim 37, wherein the composition is administered with an additionaltherapeutic agent.
 40. The composition of claim 39, wherein theadditional therapeutic agent is selected from the group consisting of achemotherapeutic agent, ablation or other therapeutic hormone,antineoplastic agent, monoclonal antibody useful against cancer, andangiogenesis inhibitor.
 41. A method of inhibiting tumor-inducedangiogenesis, comprising administering uteroglobin to a patient in needthereof.
 42. The method of claim 41, wherein the tumor is a tumor ofepithelial cell origin.
 43. The method of claim 42, wherein the tumor ofepithelial cell origin is a sarcoma.
 44. The method of claim 42, whereinthe tumor is selected from the group consisting of a breast, lung,colon, bladder, prostate, gastrointestinal track, endometrium,tracheal-bronchial tract, pancreas, liver, uterus, nasopharynges, andskin tumor.
 45. The method of claim 44, wherein the tumor is a prostatetumor.
 46. The method of claim 41, wherein the uteroglobin is humanuteroglobin.
 47. The method of claim 41, wherein the uteroglobin isadministered in combination with another treatment.
 48. The method ofclaim 47, wherein the other treatment is selected from the groupconsisting of surgical intervention, radiation therapy, hormonaltherapy, immunotherapy, chemotherapy, cryotherapy, and gene therapy. 49.The method of claim 41, wherein the uteroglobin is administered directlyto lungs of the patient.
 50. A pharmaceutical composition for inhibitingtumor-induced angiogenesis, comprising: (i) uteroglobin, and (ii) apharmaceutically acceptable carrier.
 51. The composition of claim 50,wherein the tumor is a tumor of epithelial cell origin.
 52. Thecomposition of claim 50, wherein the uteroglobin is human uteroglobin.53. The composition of claim 52, wherein the composition is administeredwith an additional therapeutic agent.
 54. The composition of claim 53,wherein the additional therapeutic agent is selected from the groupconsisting of a chemotherapeutic agent, ablation or other therapeutichormone, antineoplastic agent, monoclonal antibody useful againstcancer, and angiogenesis inhibitor.
 55. A method of inhibiting ordecreasing activity of metalloproteinases required for degradation of anextra cellular matrix to inhibit invasion of tumor cells, comprisingadministering uteroglobin to a patient in need thereof.
 56. The methodof claim 55, wherein the tumor cells are of a tumor of epithelial cellorigin.
 57. The method of claim 56, wherein the tumor is selected fromthe group consisting of a tumor of the breast, lung, colon, bladder,prostate, gastrointestinal track, endometrium, tracheal-bronchial tract,pancreas, liver, uterus, nasopharynges, and skin.
 58. The method ofclaim 57, wherein the tumor is a prostate tumor.
 59. The method of claim55, wherein the uteroglobin is human uteroglobin.
 60. The method ofclaim 55, wherein the uteroglobin is administered in combination withanother treatment.
 61. The method of claim 60, wherein the othertreatment is selected from the group consisting of surgicalintervention, radiation therapy, hormonal therapy, immunotherapy,chemotherapy, cryotherapy, and gene therapy.
 62. The method of claim 55,wherein the uteroglobin is administered directly to lungs of thepatient.
 63. A pharmaceutical composition for inhibiting or decreasingthe activity of metalloproteinases required for degradation of an extracellular matrix to inhibit invasion of tumor cells, comprising: (i)uteroglobin, and (ii) a pharmaceutically acceptable carrier.
 64. Thecomposition of claim 63, wherein the tumor cells are of a tumor ofepithelial cell origin.
 65. The composition of claim 63, wherein theuteroglobin is human uteroglobin.
 66. The composition of claim 63,wherein the composition is administered with an additional therapeuticagent.
 67. The composition of claim 66, wherein the additionaltherapeutic agent is selected from the group consisting of achemotherapeutic agent, ablation or other therapeutic hormone,antineoplastic agent, monoclonal antibody useful against cancer, andangiogenesis inhibitor.
 68. A method of treating a patient during postsurgical recovery from removal of a tumor, comprising administeringuteroglobin to a patient in need thereof.
 69. The method of claim 68,wherein the treatment lessens a chance of recurrence of the tumorengendered by shed cells that cannot be removed by surgicalintervention.
 70. The method of claim 69, wherein the treatment preventsthe shed cells from entering a lymph or circulatory system.
 71. Themethod of claim 68, wherein the tumor is a tumor of epithelial cellorigin.
 72. The method of claim 71, wherein the tumor is selected fromthe group consisting of a breast, lung, colon, bladder, prostate,gastrointestinal track, endometrium, tracheal-bronchial tract, pancreas,liver, uterus, nasopharynges, and skin tumor.
 73. The method of claim72, wherein the tumor is a prostate tumor.
 74. The method of claim 68,wherein the uteroglobin is human uteroglobin.
 75. The method of claim68, wherein the uteroglobin is administered in combination with anothertreatment.
 76. The method of claim 75, wherein the other treatment isselected from the group consisting of surgical intervention, radiationtherapy, hormonal therapy, immunotherapy, chemotherapy, cryotherapy, andgene therapy.
 77. A pharmaceutical composition for administration to apatient during post surgical recovery from removal of a tumor,comprising: (i) uteroglobin, and (ii) a pharmaceutically acceptablecarrier.
 78. The composition of claim 77, wherein the tumor is a tumorof epithelial cell origin.
 79. The composition of claim 77, wherein theuteroglobin is human uteroglobin.
 80. The composition of claim 77,wherein the composition is administered with an additional therapeuticagent.
 81. The composition of claim 80, wherein the additionaltherapeutic agent is selected from the group consisting of achemotherapeutic agent, ablation or other therapeutic hormone,antineoplastic agent, monoclonal antibody useful against cancer, andangiogenesis inhibitor.
 82. A method of preventing metastasis of aprimary tumor of epithelial cell origin, comprising administeringuteroglobin to a patient in need thereof, wherein the primary tumor isprevented from invading interstitial space of a primary tissue.
 83. Themethod of claim 82, wherein the primary tumor is prevented frompenetrating a basement membrane of the primary tissue.
 84. The method ofclaim 83, wherein the primary tumor is prevented from damaging anendothelial cell wall of a lymphatic or vascular vessel.
 85. The methodof claim 84, wherein the primary tumor is prevented from shedding cellsinto the lymphatic or vascular vessel.
 86. The method of claim 85,wherein the shed cells are prevented from surviving hemodynamic stressand host defenses present in a circulatory system of the lymphatic orvascular vessel.
 87. The method of claim 86, wherein the shed cells areprevented from lodging at a new site in the circulatory system of thelymphatic or vascular vessel.
 88. The method of claim 87, wherein theshed cells are prevented from extravasating out of the lymphatic orvascular vessel into interstitial space.
 89. The method of claim 88,wherein the shed cells are prevented from invading interstitial space ofa secondary organ.